EP3387111B1 - Microorganism strain and method for antibiotic-free, fermentative preparation of low molecular weight substances and proteins - Google Patents
Microorganism strain and method for antibiotic-free, fermentative preparation of low molecular weight substances and proteins Download PDFInfo
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- EP3387111B1 EP3387111B1 EP15816123.2A EP15816123A EP3387111B1 EP 3387111 B1 EP3387111 B1 EP 3387111B1 EP 15816123 A EP15816123 A EP 15816123A EP 3387111 B1 EP3387111 B1 EP 3387111B1
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Definitions
- the invention relates to a microorganism strain and a method for the fermentative production of low molecular weight substances and proteins without antibiotics.
- the invention is set out in the appended set of claims.
- biologicals recombinant protein pharmaceuticals
- Various microorganisms such as bacteria, yeast, filamentous fungi or even plant cells or animal cells, can be used as producers. Economically important here are inexpensive fermentation, high product yields and, in the case of proteins, correct folding or modification that leads to a functional protein.
- E. coli Due to its very well-studied genetics and physiology, the short generation time and the simple handling, the Gram-negative enterobacterium Escherichia coli ( E. coli ) is currently the most frequently used organism for the production of low-molecular substances and proteins.
- microorganisms two different microorganisms are used, the microorganisms that naturally produce the substances and those that have been genetically modified.
- the technical processes required for genetic modification have long been known in the prior art.
- the aim is to introduce the genes that are necessary for the target proteins or for the synthesis of the low molecular weight substances into the host cell. These are transcribed and translated by the host cell, modified if necessary and possibly discharged into the medium.
- the profitability of a biotechnological process depends crucially on the yields achieved by the product. These can be optimized by the expression system (host cell, genetic elements, etc.), the fermentation parameters and the nutrient media.
- the host cells can be modified in two ways.
- the new genetic information can be integrated into the genome (filamentous fungi, yeasts, etc.) and / or introduced on an extrachromosomal element (eg plasmid) (prokaryotes, yeasts, etc.).
- plasmid extrachromosomal element
- prokaryotes, yeasts, etc. With the genetic integration of the genes into the genome, they are well preserved in the host cell even without selection pressure.
- the disadvantage is that in prokaryotes only one copy of the gene is present in the host and the integration of further copies of the same gene to increase product formation via the gene dose effect is very demanding due to sequence-specific recombination events ( EP 0284126 B1 ).
- the genetic information of the target protein is usually transformed into the E. coli production strain in the form of a plasmid. Since the gene dose effect also has an effect here, the aim is to have the highest possible number of plasmid copies per cell. Since such a genetic element is easily lost from the cell due to the stress caused by both the replication of the plasmid and the production of the target protein, selection pressure must be exerted over the entire cultivation. As a standard, antibiotic resistance genes are used as selection markers, which enable the cell that has such an element to grow in the presence of antibiotics. Accordingly, only those cells that carry a plasmid can multiply. Since the loss of plasmid also means that the ability to produce the target substance or protein is lost, this has a direct effect on the yields that can be achieved in the fermentation.
- antibiotic resistance as a selection marker has been viewed increasingly critically in recent years.
- the use of antibiotics is quite expensive, especially if the resistance is based on an enzyme that breaks down antibiotics, since the antibiotic has to be continuously replenished.
- the widespread use in medicine and other areas contributes to the spread of the resistance genes to other, sometimes pathogenic, strains. This has negative consequences for the treatment of diseases.
- antibiotic-free selection systems have also been developed in the prior art.
- Several different antibiotic-free systems have been developed. These can be divided into three different basic systems. The use of auxotrophies, toxin-antitoxin systems and other procedures.
- the category of other methods includes mechanisms that do not follow any general principle, e.g. suppressor t-RNAs, fab I / triclosan (FA synthesis), operator / repressor titration ( Peubez et al. Microbial Cell Fac, 2010, 9:65 ).
- these methods are usually used for the synthesis of DNA and DNA fragments for gene therapy and are not optimized for the production of high yields of target substances.
- antibiotics instead of antibiotics, other substances such.
- Toxin-antitoxin systems consist of two genetic elements that can both be coded on the plasmid and chromosomally and on the plasmid ( Gerdes et al. Proc Natl Acad Sci USA, 1986, 83: 3116-20 ).
- the antitoxin has a neutralizing effect on the toxin. If the plasmid is lost, this mechanism and the plasmid-free one is missing Cell dies due to the chromosomally encoded or long-lived toxin.
- auxotrophic strains Another known method of selection is the complementation of auxotrophic strains.
- genes in the genome of the production strain that have essential metabolic functions are removed or inactivated. Accordingly, such genes are called essential genes.
- the resulting auxotrophic strains can only grow, multiply or survive if the metabolic function is circumvented or restored. This can be achieved both by feeding the appropriate precursors or end products of the metabolism (amino acids, bases, etc.) or by introducing the gene that has been deactivated in the host genome.
- the patent EP 0284126 B1 calls auxotrophy markers of amino acid metabolism. Since auxotrophies can easily be supplemented by adding the necessary metabolic product to the medium, these strains can be easily generated.
- the cells can also be cultivated without a plasmid in the presence of the metabolic product.
- the information for the synthesis of the amino acid / base is reintroduced into the cell through transformation and the cell can grow without supplementation. Accordingly, the selection must be made for minimal media without the supplement.
- auxotrophies as selection markers has so far not been successful, since media with a complex composition are usually used in industrial fermentation.
- waste materials are usually part of the fermentation medium, such as residues from grain (ethanol production), maize (starch production), potatoes (starch production) or yeast extract. These serve as both carbon and nitrogen sources.
- Some of these components are not precisely defined, but contain amino acids, bases, vitamins, etc., which can be absorbed from the medium. In industrial fermentation it is therefore difficult, if not impossible, to to build up sufficient selection pressure with auxotrophic strains.
- auxotrophies for the essential thymidine and D-alanine which even in complex components only occur in traces or not at all in the fermentation media (EP 0251579 A1 ; EP 0185512 B1 ). But even these systems are not suitable for efficient production in the high cell density process, which is usually aimed for. Since the cells partially die off and lyse in this process, thymidine and D-alanine or other amino acids etc. are released accordingly, which in turn can supplement the auxotrophies.
- the object of the invention is to provide a microorganism strain for the production of low molecular weight substances or proteins which remains stable even in media with complex components and in which the production plasmid is not stabilized in the cell by an antibiotic / resistance marker system.
- a microorganism strain containing in its genome a mutation in a gene which causes an auxotrophy of the strain, and a production plasmid encoding at least one enzyme for the production of a low molecular weight substance or at least one recombinant protein and a functional copy of the gene, its chromosomal Inactivation causes auxotrophy, characterized in that the auxotrophy is a non-feedable auxotrophy and it is the plsC gene or its homologous genes.
- a non-feedable auxotrophy is to be understood as meaning that the auxotrophy is growth of the microorganism cell or causes the death of the microorganism cell and cannot be supplemented by adding metabolism-specific preliminary, intermediate and / or end products to the growth medium.
- growth is reduced if the growth rate of the strain in a fermentation after the mutation of the gene is reduced to 10 10% compared to the growth rate of the strain before the mutation of the gene, preferably if there is no longer any growth.
- the mutation particularly preferably leads to the inactivation of a gene, thus causing an auxotrophy that cannot be fed and thus killing the microorganism cell.
- a gene is accordingly inactivated which is essential for an anabolic metabolic pathway necessary for the growth or survival of the cell, whereby growth or survival of the cell is not due to the addition of metabolism-specific preliminary, intermediate and / or end products in the growth medium can be reached again.
- a non-feedable auxotrophy gene within the meaning of the invention is a gene in the genome of a microorganism cell, the inactivation of which cannot be complemented by adding metabolically specific preliminary, intermediate and / or end products to the growth medium and whose inactivation leads to a reduced growth rate or death the microorganism cell leads.
- the mutation of the gene which causes the non-feedable auxotrophy of the strain preferably leads to inactivation of this gene or to inactivation of the activity of the gene product encoded by the gene.
- non-feedable auxotrophy genes examples are in Baba et al. (2006, Mol. Syst. Biol. 2: 2006.0008 ) described. According to the invention, it is plsC or its homologous genes. According to the invention, it is the plsC gene or homologous genes with the same function or activity.
- a gene within the meaning of the present invention comprises, in addition to the DNA segment that is transcribed, the DNA segments that are involved in the regulation of this copying process, that is to say the regulatory elements of the gene, such as preferably promoters and terminators.
- Homologous genes are preferably to be understood as meaning genes which code for a protein with the same activity as the protein encoded by said gene and a sequence identity greater than 30%, particularly preferably greater than 70%, to the sequences of the said genes known from databases have respective microorganism.
- the plsC gene codes for the enzyme 1-acylglycerol-3-phosphate O-acyltransferase (EC: 2.3.1.51). This enzyme transfers a fatty acid from acyl-CoA to an acylglycerol-3-phosphate, releasing a CoA-SH. The resulting diacylglycerol-3-phosphate is used for the synthesis of essential membrane components such as triglycerides and glycerophospholipids. This step is therefore necessary for the production of the membrane systems of E. coli. In E. coli can absorb fatty acids, but triglycerides and glycerophospholipids must be synthesized in the cell or in the membrane.
- the plsC gene is characterized by SEQ ID no. 3.
- plsC gene product is characterized by SEQ ID no. 4th
- plsC homologous genes are those which code for a protein with PlsC activity and which have a sequence identity greater than 30% to SEQ ID NO. 3 have. A sequence identity greater than 70% to SEQ ID no. 3. It is particularly preferably the plsC gene.
- PlsC homologues particularly preferably have a 1-acylglycerol-3-phosphate O-acyltransferase activity according to EC number 2.3.1.51 and a sequence identity greater than 70% with SEQ ID no. 4 on. It is particularly preferably the PlsC protein.
- the PlsC activity in a cell can correspond to that of Monrand et al. (1998, Biochem. Biophys. Res. Commun. 244: 79-84 ) described test.
- the degree of DNA identity is determined by the program "nucleotide blast", which can be found on the website http://blast.ncbi.nlm.nih.gov/ , which is based on the blastn algorithm.
- the preset parameters were used as algorithm parameters for aligning two or more nucleotide sequences.
- the preset parameters were used as algorithm parameters for aligning two or more protein sequences.
- a temperature-sensitive plasmid is introduced into a suitable microorganism strain which has a functional copy of a non-feedable auxotrophy gene which is to be mutated or deleted.
- the corresponding non-feedable auxotrophy gene in the genome of the strain is then inactivated.
- the temperature-sensitive plasmid in this strain is exchanged for a production plasmid at a non-permissive temperature, the production plasmid encoding at least one enzyme for the production of a low molecular weight substance or at least one recombinant protein also containing a functional copy of the non-feedable auxotrophy gene.
- strains according to the invention can be cultivated without loss of plasmid in the absence of an added selective agent or an auxotrophy compensating additive.
- any microorganism strain which has a gene whose inactivation leads to a non-feedable auxotrophy of the strain is suitable as a starting strain for producing a strain according to the invention.
- the starting strain for generating a strain according to the invention is preferably an Enterobacteriaceae strain, particularly preferably a strain of the species E scherichia coli.
- a “leaky mutation” is a mutation in a gene for a structural element of the outer cell membrane or the cell wall, selected from the group of the omp genes, tol genes, excD genes, excC genes, lpp genes, pal-
- env genes, and lky genes that lead to the cell membrane or the cell wall selected from the group of the omp genes, tol genes, excD gene, excC gene, lpp gene, pal gene, env genes and lky genes, which leads to understanding of the cells release more periplasmic proteins into the medium ( Shokri et al., Appl. Microbiol. Biotechnol.
- lpp gene is preferably a "leaky” mutation in the lpp gene, particularly preferably a mutation selected from the group lppl mutation, lpp deletion mutation and mutation of the glycine residue at position 14 of the Lpp protein (counting including signal peptide ), such as the lpp-3 mutation.
- An lppl mutation is a mutation in the lpp gene that leads to an exchange of the arginine residue at position 77 for a cysteine residue;
- an lpp3 mutation is a mutation in the lpp gene that leads to an exchange of the glycine residue leads at position 14 against an aspartic acid residue.
- the lpp deletion mutation is preferably a deletion of at least one nucleotide in the lpp gene itself or in the promoter region of the lpp gene, which leads to the cells having increased leakiness for periplasmic proteins.
- increased leakiness is to be understood as meaning that, after fermentation of the cells, a higher concentration of periplasmic proteins, e.g. B. the alkaline phosphatase, is present in the nutrient medium than in a fermentation of E. coli strain W3110 (ATCC 27325) under the same conditions.
- periplasmic proteins e.g. B. the alkaline phosphatase
- Methods for inactivating a gene in a strain of microorganisms are known in the art. They are described in detail below for the mutation of the plsC gene and its gene products. Analogously, these methods can also be used for other genes, the inactivation of which causes a non-feedable auxotrophy of a microorganism strain. formants.
- the selection marker is, for example, an antibiotic resistance.
- a preferred example of a temperature-sensitive origin of replication is "oriR101 &repA101-ts", a derivative of the origin of replication of plasmid pSC101 ( Hashimoto-Gotoh et al .; Gene, 2000, 241: 1: 185-191 ), which is found on the plasmids pKD20 and pKD46 ( Datsenko and Wanner, 2000, PNAS 97: 6640-6645 ) is localized.
- the temperature-sensitive plasmid is introduced into the cell using a transformation technique known to the person skilled in the art, e.g. TSS, CaCl / RbCl, electroporation method.
- Cells which contain the temperature-sensitive plasmid are selected by means of the selection marker which is present on the temperature-sensitive plasmid, while the cells are incubated at a temperature permissive for the plasmid.
- Such a temperature-sensitive plasmid can be exchanged for the production plasmid by cultivating the microorganism strain after the transformation with the production plasmid at a temperature which is non-permissive for the temperature-sensitive plasmid.
- a preferred non-permissive temperature range is 37-45 ° C, particularly preferably 39-43 ° C.
- the production plasmid is introduced into the cell using a transformation technique known to the person skilled in the art, e.g. TSS, CaC1 / RbCl, electroporation method.
- the transformants can be cultivated on agar plates or in liquid culture.
- the microorganism strain is exposed to a temperature shock at 47-55 ° C., preferably at 52 ° C., for 30-90 min, preferably 60 min, immediately after the transformation with the production plasmid. Further incubation then takes place at the non-permissive temperature mentioned above.
- the temperature-sensitive plasmid is exchanged for the production plasmid and a plasmid according to the invention in one step Production strain generated for the antibiotic-free production of low molecular weight substances or recombinant proteins.
- One of the following known expression vectors is used, for example, as the production plasmid: pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3 or pET.
- the production plasmid contains one or more target genes and the expression signals necessary for expressing these target genes, such as promoter, operator and terminator sequences.
- the target genes code for at least one enzyme for the production of a low molecular weight substance or at least one recombinant protein.
- the low molecular weight substances are basic building blocks (bases, amino acids, fatty acids etc.) as well as secondary metabolites (vitamins, antioxidants etc.) that the organism can synthesize. Amino acids are preferred, the amino acid L-cysteine and compounds derived therefrom are particularly preferred.
- the recombinant proteins are preferably heterologous proteins.
- a heterologous protein is understood to be a protein that does not belong to the proteome, i.e. the entire natural protein structure of the host organism.
- the heterologous protein is preferably a eukaryotic protein, particularly preferably a protein which contains one or more disulfide bridges or which is present in its functional form as a dimer or multimer, ie the protein has a quaternary structure and consists of several identical (homologous) or non-identical (heterologous) subunits.
- a preferred class of proteins that consists of several protein subunits are antibodies or fragments of antibodies. Functional Fab antibody fragments are particularly preferred.
- any host organism that has a gene for the UMP kinase PyrH is suitable as starting strains for the production of a microorganism strain with genomic pyrH inactivation.
- the pyrH gene can be inactivated, for example, by introducing a mutation (substitution, insertion or deletion of one or more nucleotides) into the reading frame of the pyrH gene, which mutation leads to the specific activity of PyrH being inactivated.
- Methods for generating such pyrH alleles are known to those skilled in the art. For example, using the in Link et al. (1997, J. Bacteriol. 179: 6228-37 ), the introduction of chromosomal mutations into a gene is carried out using the mechanism of homologous recombination.
- chromosomal deletion of the entire pyrH gene or part thereof is, for example, with the aid of the ⁇ -Red recombinase system according to that of Datsenko and Wanner (2000, Proc. Natl. Acad. Sci, USA. 97: 6640-5 ) described method possible.
- pyrH alleles can also be transferred by transduction using P1 phages or conjugation from a strain with pyrH mutation to a pyrH wild-type strain, the pyrH wild-type gene in the chromosome being replaced by the corresponding pyrH allele.
- the pyrH gene of a cell can also be inactivated by using at least one element necessary for expression regulation (e.g. promoter, enhancer, ribosome binding site) is mutated by substitution, insertion or deletion of one or more nucleotides.
- Inactivation within the meaning of the invention is when the growth rate of the cells in a fermentation is reduced to 10% compared to the cells before the mutation due to the inactivation of the gene, preferably when there is no longer any growth.
- the mutation particularly preferably leads to the death of the microorganism cell.
- a functional copy of the pyrH gene must already be present in the cell before the chromosomal inactivation. This can be achieved by the transient introduction of a temperature-sensitive plasmid containing a functional copy of the pyrH gene.
- this temperature-sensitive plasmid is exchanged for the production plasmid, which also contains a functional copy of the pyrH gene. This is preferably done in the manner already described.
- Microorganism strains according to the invention with an inactivated plsC gene can be produced analogously.
- cytoplasmic and secretory production While in cytoplasmic production the target protein is accumulated in the cytoplasm of the cell, in secretory production the target protein is translocated into the periplasm or into the culture medium. Secretory production is preferred within the scope of the invention. The secretory production of the target protein in the culture medium is particularly preferred.
- the signal sequence of the phoA or ompA gene from E. coli or the signal sequence for a cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae M5a1, or the sequence derived from this signal sequence and shown in US2008076157 is revealed.
- the cultivation (fermentation) of the cells which contain a production plasmid takes place according to conventional fermentation processes known to the person skilled in the art in a bioreactor (fermenter) without the addition of antibiotics.
- the invention also relates to a method for producing low molecular weight substances or recombinant proteins by means of a microorganism strain, which is characterized in that a microorganism strain according to the invention is used and an antibiotic-free fermentation medium is used.
- the fermentation takes place in a conventional bioreactor, for example a stirred tank, a bubble column fermenter or an airlift fermenter.
- a stirred tank fermenter on an industrial scale and thus with a size of> 100 1 is preferred.
- the cells of the strain according to the invention are cultivated in a liquid medium, with various parameters such as the nutrient supply, the oxygen partial pressure, the pH value and the temperature of the culture being continuously and precisely controlled.
- the cultivation period is preferably 16-150 hours, particularly preferably 24-72 hours.
- Complex media or minimal salt media to which a defined proportion of complex components such as peptone, tryptone, yeast extract, molasses or corn steep liquor is added can be used.
- a medium with complex media components is preferred.
- All sugars, sugar alcohols or organic acids or their salts which can be used by the cells can be used as the primary carbon source for the fermentation.
- Glucose, lactose or glycerine are preferably used.
- Glucose and lactose are particularly preferred.
- Combined feeding of several different carbon sources is also possible.
- the carbon source can be placed completely in the fermentation medium at the beginning of the fermentation or nothing or only a part of the carbon source is placed at the beginning and the carbon source is fed in over the course of the fermentation.
- An embodiment is particularly preferred in which part of the carbon source is presented and part is fed.
- the carbon source is particularly preferably provided in a concentration of 10-30 g / l, feeding is started when the concentration has fallen to less than 5 g / l and is designed so that the concentration is kept below 5 g / l.
- the oxygen partial pressure (pO 2 ) in the culture is preferably between 10 and 70% saturation.
- a pO 2 between 20 and 60% is preferred, the pO 2 is particularly preferably between 45 and 55% saturation.
- the pH value of the culture is preferably between pH 6 and pH 8. A pH value between 6.5 and 7.5 is preferred, and the pH value of the culture is particularly preferably between 6.8 and 7.2.
- the temperature of the culture is between 15 and 45 ° C. A temperature range between 18 and 40 ° C. is preferred, a temperature range between 25 and 35 ° C. is particularly preferred, and 30 ° C. is very particularly preferred.
- Example 1 Amplification of the marker gene ( pyrH or plsC ) with its own promoter
- the approximately 1.0 kb PCR fragment was purified by agarose gel electrophoresis and isolated from the agarose gel using the "QIAquick Gel Extraction Kit” (Qiagen GmbH, Hilden, D) according to the manufacturer's instructions. The purified PCR fragment was then digested with the restriction enzyme NcoI and stored at -20.degree. Similarly, an approximately 1.0 kb DNA fragment, which codes for the plsC gene including the native promoter region, was amplified, purified, digested and stored. The primers plsC-NcoI-fw (SEQ ID No. 7) and plsC-NcoI-rev (SEQ ID No. 8) were used for the amplification.
- Example 2 Generation of the plasmids pAF-ts-pyrH and pAF-ts-plsC with temperature-sensitive origin of replication
- the plasmid pKD46 was used as the starting plasmid for the construction of the plasmids pAF-ts-pyrH and pAF-ts-plsC with temperature-sensitive origin of replication ( Datsenko and Wanner, 2000, PNAS 97: 6640-6645 ).
- a restriction and function map of plasmid pKD46 is in Fig. 1 shown.
- the ligation mixtures were transformed into "DH5 ⁇ TM -T1R E.
- the plasmids pAF-ts-pyrH and pAF-ts-plsC with temperature-sensitive origin of replication described in Example 2 were converted into the two E. coli strains W3110 (ATCC 27325) and W31101pp3 (described in US2008076158 A1 as a "leaky” strain).
- the transformed cells were selected on LB agar plates which contained 100 mg / l ampicillin.
- the strains generated in this way have the designations W3110 / pAF-ts-pyrH, W31101pp3 / pAF-ts-pyrH, W3110 / pAF-ts-plsC and W31101pp3 / pAF-ts-plsC.
- Example 4 Deletion of the pyrH gene (inactivation of uridylate kinase) or of the plSC gene (inactivation of 1-acylglycerol-3-phosphate O-acyltransferase) in E. coli
- the gene pyrH which codes for the enzyme uridylate kinase (PyrH) in E. coli , was determined in the E. coli strains W3110 / pAF-ts-pyrH and W31101pp3 / pAF using the " ⁇ -Red method" developed by Datsenko and Wanner -ts-pyrH deleted ( Datsenko and Wanner, 2000, PNAS 97: 6640-6645 ).
- a DNA fragment which codes for the kanamycin resistance marker gene (kanR) was amplified with the primers pyrH-fw (SEQ ID No. 9) and pyrH-rev (SEQ ID No. 10).
- the primer pyrH-fw codes for a sequence consisting of 30 nucleotides, which is homologous to the 5 'end of the pyrH gene, and a sequence comprising 20 nucleotides that is complementary to a DNA sequence which is one of the two FRT- sites (FLP recognition target) on the plasmid pKD13 (Coli Genetic Stock Center (CGSC) No. 7633).
- the primer pyrH-rev codes for a sequence consisting of 30 nucleotides which is homologous to the 3 "end of the pyrH gene, and a sequence comprising 20 nucleotides which is complementary to a DNA sequence which is the second FRT site encoded on the plasmid pKD13.
- the amplified PCR product was introduced into the E. coli strains W3110 / pAFts-pyrH and W3110lpp3 / pAF-ts-pyrH (see Example 3) by means of electroporation.
- the selection for cells with chromosomal integration of the kanamycin resistance marker gene ( kanR ) was carried out on LB agar plates containing 50 mg / l kanamycin and 100 mg / l ampicillin.
- the chromosomally introduced kanamycin resistance marker gene ( kanR ) was removed with the enzyme FLP recombinase, which is encoded on the plasmid pCP20 (CGSC No. 7629).
- the selection for pCP20-containing cells took place on LB agar plates which contained 100 mg / l ampicillin (selection for pAF-ts-pyrH) and 34 mg / l chloramphenicol (selection for pCP20). Due to a temperature-sensitive origin of replication (ori), the plasmid pCP20 can be removed again after transformation by culturing the E. coli cells at a non-permissive, ie elevated temperature, for example at 42 ° C.
- a first selection for loss of the temperature-sensitive plasmid pCP20 with simultaneous receipt of the temperature-sensitive plasmid pAF-ts-pyrH was carried out on LB agar plates which contained 100 mg / l ampicillin (selection for pAF-ts-pyrH). Subsequently, the preselected ampicillin-resistant bacterial clones were checked for kanamycin sensitivity, i.e. the loss of the chromosomally introduced kanamycin marker gene, as well as for chloramphenicol sensitivity, i.e. the loss of the temperature-sensitive plasmid pCP20.
- the ampicillin-resistant E. coli strains with chromosomal pyrH deletion and plasmid-encoded pyrH expression have the designations W3110 ⁇ pyrH / pAF-ts-pyrH and W3110lpp3 ⁇ pyrH / pAF-ts-pyrH.
- the gene plsC which in E. coli codes for the enzyme 1-acylglycerol-3-phosphate O-acyltransferase (PlsC), was found in the E. coli strains W3110 / pAF-ts-plsC and W31101pp3 / pAF -ts-plsC deleted ( Datsenko and Wanner, 2000, PNAS 97: 6640-6645 ).
- a DNA fragment which codes for the kanamycin resistance marker gene (kanR) was amplified with the primers plsC-fw (SEQ ID No. 13) and plsC-rev (SEQ ID No. 14).
- the primer plsC-fw codes for a sequence consisting of 30 nucleotides which is homologous to the 5 'end of the plsC gene , and a sequence comprising 20 nucleotides which is complementary to a DNA sequence which is one of the two FRT- sites (FLP recognition target) on the plasmid pKD13 (Coli Genetic Stock Center (CGSC) No. 7633).
- the primer plsC-rev codes for a sequence consisting of 30 nucleotides which is homologous to the 3 'end of the plsC gene, and a sequence comprising 20 nucleotides which is complementary to a DNA sequence which is the second FRT site encoded on the plasmid pKD13.
- the amplified PCR product was introduced into the E. coli strains W3110 / pAFts-plsC and W3110lpp3 / pAF-ts-plsC (see Example 3) by means of electroporation.
- the chromosomally introduced kanamycin resistance marker gene ( kanR ) was removed again with the enzyme FLP recombinase (encoded on plasmid pCP20).
- the selection for pCP20-containing cells was also carried out here on LB agar plates containing 100 mg / l ampicillin ( Selection for pAF-ts-plsC) and 34 mg / l chloramphenicol (selection for pCP20).
- a first selection for loss of the temperature-sensitive plasmid pCP20 with simultaneous receipt of the temperature-sensitive plasmid pAF-ts-plsC was made on LB agar plates containing 100 mg / l ampicillin (selection for pAF-ts-plsC). Subsequently, the preselected ampicillin-resistant bacterial clones were checked for kanamycin sensitivity, ie the loss of the chromosomally introduced kanamycin marker gene, and for chloramphenicol sensitivity, ie the loss of the temperature-sensitive plasmid pCP20. These clones were then with the primers plsC-check-for (SEQ ID No.
- plsC-check-rev (SEQ ID No. 16) on the chromosomal deletion of the plsC gene checked.
- Chromosomal DNA of the selected ampicillin-resistant, chloramphenicol- and kanamycin-sensitive clones served as a template for checking the chromosomal plsC deletion by means of PCR.
- the ampicillin-resistant E. coli strains with chromosomal plsC deletion and plasmid-encoded plsC expression have the designations W3110 ⁇ plsC / pAF-ts-plsC and W3110lpp3 ⁇ plsC / pAF-ts-plsC.
- Example 5 Generation of a production plasmid with antibiotic resistance genes for the production of cysteine
- the basic plasmid pMT1 and the in. Were used as starting plasmids for the cloning and expression of the genes cysEX (encoded for feedback-resistant variant of serine acyltransferase; CysE) and orf306 (encoded for O-acetylserine / cysteine exporter; EamA)
- EP0885962B1 described production plasmid pACYC184-LH-cysEX-orf306.
- pMT1 In addition to the tetracycline resistance gene (tetR), pMT1 also contains the tac promoter, which is repressed by the LacIq gene product, the gene of which is also present on the plasmid, and which is produced by an inducer such as D-lactose or isopropyl- ⁇ -D- thiogalactopyranoside (IPTG) can be switched on.
- an inducer such as D-lactose or isopropyl- ⁇ -D- thiogalactopyranoside (IPTG)
- a restriction and function map of plasmid pMT1 is in Fig. 4 shown.
- the sequence of the plasmid pMT1 is deposited in the sequence listing (SEQ ID No. 17).
- an NcoI-BsaBI fragment of the plasmid pACYC184-LH-cysEX-orf306 (described in EP0885962 B1 ), which codes for the genes cysEX and orf306, ligated with a 2458 bp NcoI-PvuII fragment (codes for ColE1-ori and tetracycline resistance, tetR) of the plasmid pMT1.
- the ligation mixture was transformed into "DH5 ⁇ TM -T1R E. coli cells" (Life Technologies GmbH), propagated in these cells and the DNA sequence of the isolated plasmids was verified by means of sequencing.
- the resulting expression plasmid has the designation pcysEX-GAPDH-ORF306_tetR (see Fig. 5 ).
- Example 6 Generation of a production plasmid with antibiotic resistance genes for the production of ⁇ -CGTase
- the starting plasmids for the cloning and expression of the cyclodextrin glycosyl transferase (CGTase) gene from Klebsiella pneumoniae M5al were again the plasmid pMT1 and the in US2008076158 A1 plasmid pCGT described.
- CGTase cyclodextrin glycosyl transferase
- a MauBI-BsaI fragment of the plasmid pCGT which codes for the CGTase gene from Klebsiella pneumoniae M5a1, with a 4004 bp MauBI-BsaI fragment of the plasmid pMT1 ligated.
- This 4004 bp fragment of the plasmid pMT1 codes for the ColE1-ori, the lac / tac operator and the tetracycline resistance gene ( tetR ).
- the ligation mixture was transformed into "DH5 ⁇ TM -T1R E. coli cells" (Life Technologies GmbH), propagated in these cells and the DNA sequence of the isolated plasmids was verified by means of sequencing.
- the resulting expression plasmid has the designation pCGT_tetR (see Fig. 6 ).
- Example 7 Generation of a production plasmid with antibiotic resistance genes for the production of Fab-anti-lysozyme
- the plasmid pMT1 and the in were again used as starting plasmids for the cloning and expression of the genes of the anti-lysozyme Fab fragment US20080076158 A1 described pFab-anti-lysozyme.
- a MauBI-BsaI fragment of the plasmid Fab-anti-lysozyme which is responsible for the two chains, ie the heavy chain (V E -C H 1 domains) and the light chain (V L -C L domains) of the anti-lysozyme Fab fragment, ligated with a 4004 bp MauBI-BsaI fragment of the plasmid pMT1.
- This 4004 bp fragment of the plasmid pMT1 codes for the ColE1-ori, the lac / tac operator and the tetracycline resistance gene ( tetR ).
- the ligation mixture was transformed into "DH5 ⁇ TM -T1R E. coli cells" (Life Technologies GmbH), and multiplied in these cells Fragment of the plasmid pMT1 codes for the ColE1-ori, the lac / tac operator and the tetracycline resistance gene ( tetR ).
- the ligation mixture was transformed into "DH5 ⁇ TM -T1R E. coli cells" (Life Technologies GmbH), propagated in these cells and the DNA sequence of the isolated plasmids was verified by means of sequencing.
- the resulting expression plasmid has the designation pFab-anti-Lysozyme_tetR (see Fig. 7 ).
- Example 8 Generation of production plasmids with the marker genes pyrH or plsC as the basis for creating production plasmids according to the invention without an antibiotic resistance gene
- Example 9 Removal of the antibiotic resistance gene tetR and transformation of the production plasmids with pyrH or plsC as the remaining marker gene in E. coli strains with chromosomal pyrH or plsC deletion
- the antibiotic resistance gene tetR was removed from the plasmids pFab-anti-Lysozyme_pyrH1_tetR and pFab-anti-Lysozyme_plsC1_tetR described in Example 8 via digestion with the restriction enzyme ClaI and subsequent religation.
- the tetR gene was removed from the plasmid by digestion with the enzymes StuI (cuts blunt end) and FspI (cuts blunt end).
- the tetR gene was also removed by partial digestion with the enzymes StuI (cuts blunt end) and FspI (cuts blunt end), since there is another FspI cleavage site in the pyrH gene (see FIG Fig. 9 ).
- the respective linear vector fragments without tetR were purified after restriction digestion via agarose gel electrophoresis and extracted with the “QIAquick Gel Extraction Kit” (Qiagen and FspI (cuts blunt end) removed because another FspI cleavage site is located in the pyrH gene (see Fig. 9 ).
- the corresponding ligation batches were converted into the strains W3110 ⁇ pyrH / pAF-ts-pyrH and W31101pp3 ⁇ pyrH / pAF-ts-pyrH or W3110 ⁇ pyrH / pAF-ts-plsC or W31101pp3 ⁇ pyrs- / pAF-tsC described in Example 4 using a modified CaCl 2 method plsC transformed.
- a modified CaCl 2 method plsC For the transformation, ie the introduction of the antibiotic resistance-free production plasmids with pyrH or plsC as marker gene in E.
- coli strains with a corresponding chromosomal deletion ( pyrH or plsC )
- the following procedure was used: After adding 5 to 20 ⁇ l of the respective ligation batch to 100 ⁇ l CaCl 2 -competent cells of the strains W3110 ⁇ pyrH / pAF-ts-pyrH and W3110lpp3 ⁇ pyrH / pAF-ts-pyrH or W3110 ⁇ plsC / pAF-ts-plsC and W3110lpp3 ⁇ plsC and With ts-plsC, the cells were incubated on ice for a further 30 minutes. After a brief heat shock for 45 seconds at 42 ° C., the cells were cooled on ice for 2 minutes.
- the 30-90 minute regeneration phase at 47 ° C to 55 ° C and cultivation for 15 to 24 h at an elevated, non-permissive temperature facilitate the exchange of the temperature-sensitive plasmids pAF-ts-pyrH or pAF-ts-plsC for the final, Antibiotic resistance-free production plasmid with pyrH or plsC as a new selection marker gene.
- the preselected bacterial clones were then checked for ampicillin sensitivity, i.e. the loss of the temperature-sensitive plasmid pAF-ts-pyrH or pAF-ts-plsC.
- the production plasmids encoding pyrH or plsC from ampicillin-sensitive clones were then checked by means of restriction digestion. Restriction maps of the pyrH -coding production plasmids pcysEX-GAPDH-ORF306_pyrH, pCGT_pyrH and pFab-anti-Lysozyme_pyrH, each without the antibiotic resistance gene tetR , are shown in FIGS. 14 to 16.
- the preculture 1 was completely transferred into 100 ml of SM1 medium (12 g / l K 2 HPO4, 3 g / l KH 2 PO 4 , 5 g / l (NH 4 ) 2SO 4 , 0.3 g / l MgSO 4 x 7 H 2 O, 0.015 g / l CaCl 2 x 2 H 2 O, 0.002 g / l FeSO 4 x 7 H 2 O, 1 g / l Na 2 citrate x 2 H 2 O, 0.1 g / l NaCl , 1 ml / l trace element solution, consisting of 0.15 g / l Na 2 MoO 4 x 2H 2 O, 2.5 g / l H 3 BO 3 , 0.7 g / l CoCl 2 x 6 H 2 O, 0 , 25 g / l CuSO 4 x 5 H 2 O, 1.6 g / l MnCl
- the fermentation was carried out in fermenters of the type BIOSTAT B from Sartorius Stedim. A culture vessel with a total volume of 2 liters was used.
- the fermentation medium (900 ml) contains 15 g / l glucose, 10 g / l tryptone (Difco), 5 g / l yeast extract (Difco), 5 g / l (NH 4 ) 2 SO 4 , 1.5 g / l KH 2 PO 4 , 0.5 g / l NaCl, 0.3 g / l MgSO 4 x 7 H 2 O, 0.015 g / l CaCl 2 x 2 H 2 O, 0.075 g / l FeSO 4 x 7 H 2 O, 1 g / l Na 2 citrate x 2 H 2 O and 1 ml trace element solution (see above) and 0.005 g / l vitamin B1.
- the pH in the fermenter was initially adjusted to 6.5 by pumping in a 25% NH4OH solution. During the fermentation, the pH was kept at a value of 6.5 by automatic correction with 25% NH 4 OH.
- 100 ml of preculture 2 were pumped into the fermenter vessel. The initial volume was thus about 11.
- the cultures were initially stirred at 400 rpm and gassed with 2 vvm of compressed air disinfected via a sterile filter. Under these starting conditions, the oxygen probe was calibrated to 100% saturation before the inoculation.
- the target value for the O 2 saturation during the fermentation was set to 50%. After the O 2 saturation had fallen below the target value, a regulation cascade was started in order to bring the O 2 saturation back to the target value. First the Gas supply increased continuously (to max. 5 vvm) and then the stirring speed (to max. 1,500 rpm) increased continuously.
- the fermentation was carried out at a temperature of 30 ° C.
- a sulfur source was fed in in the form of a sterile 60% sodium thiosulfate x 5 H 2 O stock solution at a rate of 1.5 ml per hour.
- a 56% glucose solution was continuously metered in.
- the feeding rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g / l.
- the glucose determination was carried out with a glucose analyzer from YSI (Yellow Springs, Ohio, USA).
- a glucose analyzer from YSI (Yellow Springs, Ohio, USA).
- the medium was supplemented with 15 mg / L tetracycline.
- the fermentation time was 48 hours. Samples were then taken and the content of L-cysteine and the derivatives derived therefrom in the culture supernatant (especially L-cysteine and thiazolidine) and in the precipitate (L-cystine) were determined separately from one another.
- the Gaitonde colorimetric test was used for this purpose ( Gaitonde, MK (1967), Biochem. J. 104, 627-633 ).
- the L-cystine in the precipitate first had to be dissolved in 8% hydrochloric acid before it could be quantified in the same way.
- the values for total cysteine listed in Table 1 correspond to the sum of the L-cysteine in the culture supernatant and L-cystine in the precipitate.
- Each L-cystine molecule corresponds to two L-cysteine molecules.
- Table 1 Total cysteine content (L-cysteine ⁇ sub> culture supernatant ⁇ /sub> + L-cystine ⁇ sub> precipitate ⁇ /sub>) in the culture broth after 48 h and stability of the production plasmids ⁇ /b> tribe Total cysteine (g / L) Plasmid stability
- Example 11 Secretory production of a cyclodextrin glycosyl transferase on a 10 1 scale (fermentation)
- biotechnologically relevant enzymes such as CGTases can be produced and secreted into the medium ( US2008076158 A1 ).
- the one with 6 l of fermentation medium FM4 (1.5 g / l KH 2 PO 4 ; 5 g / l (NH 4 ) 2 SO 4 ; 0.5 g / l MgSO 4 x 7 H 2 O; 0.15 g / l CaCl 2 x 2 H 2 O, 0.075 g / l FeSO 4 x 7 H 2 O; 1 g / l Na 2 citrate x 2 H 2 O; 0.5 g / l NaCl; 1 ml / 1 trace element solution (0, 15 g / l Na 2 MoO 4 x 2 H 2 O; 2.5 g / l Na 3 BO 3 ; 0.7 g / l CoCl 2 x 6 H 2 O; 0.25 g / l CuSO 4 x 5 H 2 O; 1.6 g / l MnCl 2 x 4 H 2 O; 0.3 g / l ZnSO 4 x 7 H 2 O); 5 mg /
- Table 2 lists the yields of functional CGTase and the activities in the fermentation supernatant.
- Table 2 Cyclodextrin glycosyltransferase yields in the fermentation supernatant after 72 hours of fermentation ⁇ /b> tribe CGTase (U / ml) CGTase (mg / l) Plasmid stability W3110lpp3 / pCGT_tetR (cultivated with tetracycline) * 555 ⁇ 15 3750 ⁇ 145 97% ⁇ 3% W3110lpp3 / pCGT_tetR (cultivated without tetracycline) 340 ⁇ 65 2210 ⁇ 425 55% ⁇ 21% W3110lpp3 ⁇ pyrH / pCGT pyrH ** 560 + 25 3795 + 160 98% + 2% W3110lpp3 ⁇ plsC / pCGT_plsC ** 570 + 30 3810 + 190 98% + 2% *
- Example 12 Secretory, fermentative production of the Fab antibody fragment anti-lysozyme Fab on a 10 l scale
- the present example describes the production of a Fab fragment of the well characterized anti-lysozyme antibody D1.3.
- the plasmids pFab-anti-Lysozym_tetR, pFab-anti-Lysozym_pyrH and pFab-anti-Lysozym_plsC contain not only the marker genes tetR, pyrH or plsC , but also the structural genes for the HC and LC of the Fab fragment in the form of an operon.
- the HC in frame is fused to the 3 'end of the ompA signal sequence (ompA ss ) and the LC in frame to the 3' end of a CGTase signal sequence (cgt ss ).
- the expression of the ompA ss -HC-cgt ss -LC operon is under the control of the tac promoter.
- the anti-lysozyme Fab fragment was produced on a 10 l scale analogously to the method described in Example 11 using CGTase with the strains W3110lpp3 / pFab-anti-lysozyme_tetR, W3110lpp3 ⁇ pyrH / pFab-anti-Lysozyme_pyrH and W3110lpp3 ⁇ plsC / pFab-anti-pFab -Lysozyme_plsC.
- the medium was supplemented with 15 mg / L tetracycline.
- the anti-lysozyme Fab fragment was purified from the fermentation supernatants by means of affinity chromatography as in FIG Skerra (1994, Gene 141, 79-84 ) described.
- the quantification and the determination of the activity of the purified anti-lysozyme Fab fragment was carried out using an ELISA test with lysozyme as the antigen ( Skerra, 1994, Gene 141, 79-84 ).
- Table 2 lists the extrapolated yields of functional anti-lysozyme Fab fragment in the fermentation supernatant, based on isolated amounts from each 20 ml fermentation supernatant after 72 h fermentation.
- the quantification and the determination of the activity of the purified anti-lysozyme Fab fragment was carried out using an ELISA test with lysozyme as the antigen ( Skerra, 1994, Gene 141, 79-84 ).
- Table 2 lists the extrapolated yields of functional anti-lysozyme Fab fragment in the fermentation supernatant, based on isolated amounts from each 20 ml fermentation supernatant after 72 h fermentation.
- Table 3 Anti-lysozyme Fab fragment yields in the fermentation supernatant after 72 h fermentation ⁇ /b> tribe Anti-lysozyme Fab fragment yield [mg / l] in the fermentation supernatant (extrapolated) Plasmid stability W3110lpp3 / pFab-anti-Lysozym_tetR (cultivated with tetracycline) * 1440 + 110 97% ⁇ 3% W3110lpp3 / pFab-anti-Lysozym_tetR (cultivated without tetracycline) 625 + 250 55% + 20% W3110lpp3 ⁇ pyrH / pFab-anti-lysozyme_pyrH ** 1580 + 115 98% + 2% W3110lpp3 ⁇ ⁇ pyr
- the plasmid stability was checked by means of plasmid preparation with subsequent restriction digestion. For this purpose, after the cultivation of the production strains had ended (eg after 72 h fermentation), various dilutions of the cultures were plated out on LB agar plates. For the subsequent determination of the plasmid stability, ie the identification of plasmid-carrying Cells (colonies) were only used LB plates with single colonies for the evaluation.
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Description
Die Erfindung betrifft einen Mikroorganismenstamm und ein Verfahren zur antibiotikafreien fermentativen Herstellung von niedermolekularen Substanzen und Proteinen. Die Erfindung ist in dem beigefügten Anspruchssatz aufgeführt.The invention relates to a microorganism strain and a method for the fermentative production of low molecular weight substances and proteins without antibiotics. The invention is set out in the appended set of claims.
Prinzipiell hat die natürliche Produktion von niedermolekularen Substanzen mit Hilfe von Mikroorganismen zugenommen und auch der Markt für rekombinante Proteinpharmazeutika ("Biologics") ist in den letzten Jahren stark gewachsen. Aufgrund des starken Kostendrucks bei der fermentativen Produktion, insbesondere für Pharmawirkstoffe auf Proteinbasis, wird laufend nach effizienteren und damit kostengünstigeren Verfahren und Systemen für deren Herstellung gesucht. Als Produzenten können verschiedene Mikroorganismen, wie Bakterien, Hefen, filamentöse Pilze oder auch Pflanzenzellen oder Tierzellen verwendet werden. Wirtschaftlich ausschlaggebend ist hierbei eine kostengünstige Fermentation, hohe Produktausbeuten und bei Proteinen eine korrekte Faltung bzw. Modifikation, die zu einem funktionellen Protein führt. Aufgrund seiner sehr gut untersuchten Genetik und Physiologie, der kurzen Generationszeit und der einfachen Handhabung, ist das Gram-negative Enterobakterium Escherichia coli (E. coli) gegenwärtig der am häufigsten verwendete Organismus zur Produktion niedermolekularer Substanzen und Proteine.In principle, the natural production of low molecular weight substances with the help of microorganisms has increased and the market for recombinant protein pharmaceuticals ("biologics") has also grown rapidly in recent years. Due to the strong cost pressure in fermentative production, especially for pharmaceutical active ingredients based on protein, more efficient and thus more cost-effective processes and systems for their production are constantly being sought. Various microorganisms, such as bacteria, yeast, filamentous fungi or even plant cells or animal cells, can be used as producers. Economically important here are inexpensive fermentation, high product yields and, in the case of proteins, correct folding or modification that leads to a functional protein. Due to its very well-studied genetics and physiology, the short generation time and the simple handling, the Gram-negative enterobacterium Escherichia coli ( E. coli ) is currently the most frequently used organism for the production of low-molecular substances and proteins.
Grundsätzlich werden zwei unterschiedliche Mikroorganismen verwendet, die Mikroorganismen, die die Substanzen natürlicherweise produzieren und solche, die genetisch modifiziert wurden. Die zur genetischen Modifikation benötigten technischen Verfahren sind im Stand der Technik seit langem bekannt. Ziel ist es dabei die Gene, die für die Zielproteine bzw. zur Synthese der niedermolekularen Substanzen notwendig sind in die Wirtszelle einzubringen. Diese werden von der Wirtszelle transkribiert, translatiert, soweit nötig modifiziert und eventuell in das Medium ausgeschleust.Basically, two different microorganisms are used, the microorganisms that naturally produce the substances and those that have been genetically modified. The technical processes required for genetic modification have long been known in the prior art. The aim is to introduce the genes that are necessary for the target proteins or for the synthesis of the low molecular weight substances into the host cell. These are transcribed and translated by the host cell, modified if necessary and possibly discharged into the medium.
Die Wirtschaftlichkeit eines biotechnologischen Verfahrens hängt entscheidend von den erzielten Ausbeuten des Produktes ab. Diese können durch das Expressionssystem (Wirtszelle, genetische Elemente etc.), den Fermentationsparametern und den Nährmedien optimiert werden.The profitability of a biotechnological process depends crucially on the yields achieved by the product. These can be optimized by the expression system (host cell, genetic elements, etc.), the fermentation parameters and the nutrient media.
Grundlegend können die Wirtszellen in zweierlei Weise modifiziert werden. So kann die neue genetische Information ins Genom integriert werden (filamentöse Pilze, Hefen, etc.) und/oder auf einem extrachromosomalen Element (z.B. Plasmid) eingebracht werden (Prokaryonten, Hefen etc.). Bei der genetischen Integration der Gene ins Genom bleiben diese auch ohne Selektionsdruck gut in der Wirtszelle erhalten. Nachteilig ist aber, dass bei Prokaryonten nur eine Kopie des Gens im Wirt vorhanden ist und die Integration weiterer Kopien desselben Gens zur Steigerung der Produktbildung über den Gendosiseffekt, aufgrund von sequenzspezifischen Rekombinationsereignissen sehr anspruchsvoll ist (
Bei der Nutzung extrachromosomaler DNA wird in der Regel die genetische Information des Zielproteins in Form eines Plasmids in den E. coli Produktionsstamm transformiert. Da sich auch hier der Gendosis-Effekt auswirkt, wird eine möglichst hohe Anzahl an Plasmidkopien pro Zelle angestrebt. Da ein solches genetische Element aufgrund der Belastung, sowohl durch die Replikation des Plasmids als auch durch die Produktion des Zielproteins, leicht aus der Zelle verloren geht, muss über die gesamte Kultivierung ein Selektionsdruck ausgeübt werden. Standardmäßig werden als Selektionsmarker Antibiotika-Resistenzgene verwendet, die damit der Zelle, die ein solches Element aufweist, ein Wachstum in Gegenwart von Antibiotika ermöglicht. Entsprechend können sich nur die Zellen vermehren, die ein Plasmid tragen. Da durch den Plasmidverlust auch die Fähigkeit zur Produktion der Zielsubstanz bzw. des Zielproteins verloren geht, hat dies einen direkten Effekt auf die Ausbeuten, die in der Fermentation erzielt werden können.When using extrachromosomal DNA, the genetic information of the target protein is usually transformed into the E. coli production strain in the form of a plasmid. Since the gene dose effect also has an effect here, the aim is to have the highest possible number of plasmid copies per cell. Since such a genetic element is easily lost from the cell due to the stress caused by both the replication of the plasmid and the production of the target protein, selection pressure must be exerted over the entire cultivation. As a standard, antibiotic resistance genes are used as selection markers, which enable the cell that has such an element to grow in the presence of antibiotics. Accordingly, only those cells that carry a plasmid can multiply. Since the loss of plasmid also means that the ability to produce the target substance or protein is lost, this has a direct effect on the yields that can be achieved in the fermentation.
Der Einsatz von Antibiotikaresistenzen als Selektionsmarker wird in den letzten Jahren zunehmend kritisch betrachtet. Zum einen ist der Einsatz von Antibiotika recht teuer, insbesondere, wenn die Resistenz auf einem Antibiotika abbauenden Enzym beruht, da das Antibiotikum stetig nachdosiert werden muss. Andererseits trägt der weitverbreitete Einsatz in der Medizin und anderen Gebieten zur Ausbreitung der Resistenzgene auf andere, teilweise pathogene Stämme bei. Dies hat negative Folgen für die Behandlung von Krankheiten.The use of antibiotic resistance as a selection marker has been viewed increasingly critically in recent years. On the one hand, the use of antibiotics is quite expensive, especially if the resistance is based on an enzyme that breaks down antibiotics, since the antibiotic has to be continuously replenished. On the other hand, the widespread use in medicine and other areas contributes to the spread of the resistance genes to other, sometimes pathogenic, strains. This has negative consequences for the treatment of diseases.
Im Stand der Technik sind inzwischen auch antibiotikafreie Selektionssysteme entwickelt worden. Mehrere unterschiedliche antibiotikafreie Systeme wurden entwickelt. Diese können in drei unterschiedliche Grundsysteme unterteilt werden. Die Verwendung von Auxotrophien, Toxin-Antitoxin-Systemen und weiteren Verfahren.In the meantime, antibiotic-free selection systems have also been developed in the prior art. Several different antibiotic-free systems have been developed. These can be divided into three different basic systems. The use of auxotrophies, toxin-antitoxin systems and other procedures.
In die Kategorie der weiteren Verfahren fallen Mechanismen, die keinem allgemeinen Prinzip folgen, z.B. Suppressor t-RNAs, fab I/Triclosan (FA-Synthese), Operator/Repressor Titration (
Toxin-Antitoxin-Systeme (Hok-Sok, ccdA/B etc.) bestehen aus zwei genetischen Elementen, die sowohl beide auf dem Plasmid, als auch chromosomal und auf dem Plasmid kodiert sein können (
Eine weitere bekannte Methode zur Selektion ist die Komplementierung auxotropher Stämme. Hier werden Gene im Genom des Produktionsstammes entfernt bzw. inaktiviert, die essentielle Funktionen im Stoffwechsel haben. Entsprechend werden solche Gene als essentielle Gene bezeichnet. Die somit entstandenen auxotrophen Stämme können nur dann wachsen, sich vermehren oder überleben, wenn die Stoffwechselfunktion umgangen oder wieder hergestellt wird. Dies kann sowohl durch Fütterung entsprechender Vorstufen oder Endprodukte des Stoffwechsels (Aminosäuren, Basen etc.) oder durch Einbringen des Gens, das im Wirtsgenom deaktiviert wurde, erreicht werden. Das Patent
In der Praxis konnte sich der Einsatz solcher Auxotrophien als Selektionsmarker bisher nicht durchsetzen, da in der industriellen Fermentation üblicherweise Medien mit komplexer Zusammensetzung verwendet werden. Hier sind in der Regel aus Kostengründen Abfallstoffe Bestandteil des Fermentationsmediums, wie z.B. Rückstände von Getreide (Ethanolherstellung), Mais (Stärkeherstellung), Kartoffeln (Stärkegewinnung) oder Hefeextrakt. Diese dienen sowohl als Kohlenstoff- als auch als Stickstoffquellen. Teilweise sind diese Bestandteile nicht genau definiert, enthalten aber Aminosäuren, Basen, Vitamine etc., die aus dem Medium aufgenommen werden können. In der industriellen Fermentation ist es daher schwer, wenn nicht unmöglich, einen ausreichenden Selektionsdruck mit auxotrophen Stämmen aufzubauen.In practice, the use of such auxotrophies as selection markers has so far not been successful, since media with a complex composition are usually used in industrial fermentation. For cost reasons, waste materials are usually part of the fermentation medium, such as residues from grain (ethanol production), maize (starch production), potatoes (starch production) or yeast extract. These serve as both carbon and nitrogen sources. Some of these components are not precisely defined, but contain amino acids, bases, vitamins, etc., which can be absorbed from the medium. In industrial fermentation it is therefore difficult, if not impossible, to to build up sufficient selection pressure with auxotrophic strains.
Auch die Verwendung einer Auxotrophie im Glukosestoffwechsel, wie sie in
Dies gilt auch für die in
Ausnahmen stellen Auxotrophien für das essentielle Thymidin und D-Alanin dar, die auch in komplexen Bestandteilen lediglich in Spuren oder gar nicht in den Fermentationsmedien vorkommen (
Insgesamt muss festgestellt werden, dass trotz jahrelanger Erfahrung zur fermentativen Produktion von niedermolekularen Substanzen und Proteinen bisher kein universell einsetzbares System entwickelt wurde, außer jenem über teure oder gesundheitlich bzw. ökologisch bedenkliche Substanzen, wie Antibio-System entwickelt wurde, außer jenem über teure oder gesundheitlich bzw. ökologisch bedenkliche Substanzen, wie Antibiotika. Auch die verschiedenen Ansätze zur Selektion über Auxotrophiemarker haben wegen der in der Industrie verwendeten komplexen Wachstumsmedien bisher nur zu dürftigen Ergebnissen geführt.Overall, it must be stated that despite years of experience in the fermentative production of low molecular weight substances and proteins, no universally applicable system has yet been developed, apart from that using expensive or health or ecologically questionable substances, such as the antibiotic system was developed, except for the one about expensive or health or ecological questionable substances such as antibiotics. The various approaches to selection via auxotrophy markers have so far only led to poor results because of the complex growth media used in industry.
Dies gilt prinzipiell für Mikroorganismen, aber insbesondere für weniger robuste Produktionsstämme, wie z.B. leaky Stämme, die aufgrund ihrer speziellen Eigenschaften (Freisetzung von Proteinen ins Medium) genutzt werden. Bei der industriellen Nutzung dieser Stämme im technischen Maßstab werden üblicherweise komplexe Bestandteile im Medium verwendet.This applies in principle to microorganisms, but especially to less robust production strains, such as leaky strains, which are used due to their special properties (release of proteins into the medium). When these strains are used industrially on an industrial scale, complex components are usually used in the medium.
Die Nutzung definierter Medien aus gereinigten Bestandteilen ist für die Herstellung von industriell genutzten Produkten aus Kostengründen nicht wirtschaftlich.The use of defined media from purified components is not economical for the production of industrially used products for reasons of cost.
Aufgabe der Erfindung ist es, einen Mikroorganismenstamm zur Produktion von niedermolekularen Substanzen oder Proteinen bereitzustellen, der auch in Medien mit Komplexbestandteilen stabil bleibt und bei dem das Produktionsplasmid nicht durch ein Antibiotikum/Resistenzmarker-System in der Zelle stabilisiert wird.The object of the invention is to provide a microorganism strain for the production of low molecular weight substances or proteins which remains stable even in media with complex components and in which the production plasmid is not stabilized in the cell by an antibiotic / resistance marker system.
Diese Aufgabe wird gelöst durch einen Mikroorganismenstamm enthaltend in seinem Genom eine Mutation in einem Gen, welche eine Auxotrophie des Stammes bewirkt, sowie ein Produktionsplasmid kodierend mindestens ein Enzym zur Produktion einer niedermolekularen Substanz oder mindestens ein rekombinantes Protein sowie eine funktionelle Kopie des Gens, dessen chromosomale Inaktivierung die Auxotrophie bewirkt, dadurch gekennzeichnet, dass es sich bei der Auxotrophie um eine nicht fütterbare Auxotrophie handelt und es sich um das plsC Gen oder dessen homologe Gene handelt.This object is achieved by a microorganism strain containing in its genome a mutation in a gene which causes an auxotrophy of the strain, and a production plasmid encoding at least one enzyme for the production of a low molecular weight substance or at least one recombinant protein and a functional copy of the gene, its chromosomal Inactivation causes auxotrophy, characterized in that the auxotrophy is a non-feedable auxotrophy and it is the plsC gene or its homologous genes.
Im Rahmen der Erfindung ist unter einer nicht fütterbaren Auxotrophie zu verstehen, dass die Auxotrophie das Wachstum der Mikroorganismenzelle vermindert oder den Tod der Mikroorganismenzelle bewirkt und nicht durch Zugabe von stoffwechselspezifischen Vor-, Zwischen- und/oder Endprodukten in das Wachstumsmedium supplementierbar ist.In the context of the invention, a non-feedable auxotrophy is to be understood as meaning that the auxotrophy is growth of the microorganism cell or causes the death of the microorganism cell and cannot be supplemented by adding metabolism-specific preliminary, intermediate and / or end products to the growth medium.
Ein vermindertes Wachstum liegt im Sinne der Erfindung vor, wenn die Wachstumsrate des Stammes in einer Fermentation nach der Mutation des Gens im Vergleich zu der Wachstumsrate des Stammes vor der Mutation des Gens auf ≤ 10% erniedrigt ist, bevorzugt wenn kein Wachstum mehr stattfindet.
Besonders bevorzugt führt die Mutation zur Inaktivierung eines Gens, bewirkt so eine nicht fütterbaren Auxotrophie und, damit den Tod der Mikroorganismenzelle.
Im Genom einer Zelle des erfindungsgemäßen Mikroorganismenstammes ist demnach ein Gen inaktiviert, welches essentiell für einen für das Wachstum oder Überleben der Zelle notwendigen anabolen Stoffwechselweg ist, wobei Wachstum oder Überleben der Zelle nicht durch Zugabe von stoffwechselspezifischen Vor-, Zwischen- und/oder Endprodukten in das Wachstumsmedium wieder erreicht werden kann.For the purposes of the invention, growth is reduced if the growth rate of the strain in a fermentation after the mutation of the gene is reduced to 10 10% compared to the growth rate of the strain before the mutation of the gene, preferably if there is no longer any growth.
The mutation particularly preferably leads to the inactivation of a gene, thus causing an auxotrophy that cannot be fed and thus killing the microorganism cell.
In the genome of a cell of the microorganism strain according to the invention, a gene is accordingly inactivated which is essential for an anabolic metabolic pathway necessary for the growth or survival of the cell, whereby growth or survival of the cell is not due to the addition of metabolism-specific preliminary, intermediate and / or end products in the growth medium can be reached again.
Ein nicht fütterbares Auxotrophie-Gen im Sinne der Erfindung ist ein Gen im Genom einer Mikroorganismenzelle, dessen Inaktivierung nicht durch Zugabe von stoffwechselspezifischen Vor-, Zwischen- und/oder Endprodukten in das Wachstumsmedium komplementiert werden kann und dessen Inaktivierung zu einer verminderten Wachstumsrate oder zum Tod der Mikroorganismenzelle führt.A non-feedable auxotrophy gene within the meaning of the invention is a gene in the genome of a microorganism cell, the inactivation of which cannot be complemented by adding metabolically specific preliminary, intermediate and / or end products to the growth medium and whose inactivation leads to a reduced growth rate or death the microorganism cell leads.
Vorzugsweise führt die Mutation des Gens, welche die nicht fütterbare Auxotrophie des Stammes bewirkt, zur Inaktivierung dieses Gens oder zur Inaktivierung der Aktivität des durch das Gen kodierten Genprodukts.The mutation of the gene which causes the non-feedable auxotrophy of the strain preferably leads to inactivation of this gene or to inactivation of the activity of the gene product encoded by the gene.
Beispiele für nicht fütterbare Auxotrophie-Gene (lethale Gene) sind in
Ein Gen im Sinn der vorliegenden Erfindung umfasst neben dem DNA Abschnitt der transkribiert wird auch die DNA Abschnitte, die an der Regulation dieses Kopiervorgangs beteiligt sind, also die regulatorischen Elemente des Gens, wie bevorzugt Promotoren und Terminatoren.A gene within the meaning of the present invention comprises, in addition to the DNA segment that is transcribed, the DNA segments that are involved in the regulation of this copying process, that is to say the regulatory elements of the gene, such as preferably promoters and terminators.
Unter homologen Genen sind vorzugsweise Gene zu verstehen, die für ein Protein mit der gleichen Aktivität kodieren wie das durch das genannte Gen kodierte Protein und eine Sequenzidentität größer 30%, besonders bevorzugt größer 70%, zu den jeweils aus Datenbanken bekannten Sequenzen der genannten Gene im jeweiligen Mikroorganismus aufweisen.Homologous genes are preferably to be understood as meaning genes which code for a protein with the same activity as the protein encoded by said gene and a sequence identity greater than 30%, particularly preferably greater than 70%, to the sequences of the said genes known from databases have respective microorganism.
Das plsC-Gen kodiert für das Enzym 1-Acylglycerol-3-Phosphat O-Acyltransferase (EC: 2.3.1.51). Dieses Enzym überträgt eine Fettsäure von Acyl-CoA auf ein Acylglycerol-3-Phosphat unter Freisetzung eines CoA-SH. Das entstehende Diacylglycerol- 3-Phosphat wird zur Synthese essentiellen Membranbestandteilen wie Triglyceriden und Glycerophospholipiden verwendet. Damit ist dieser Schritt für die Produktion der Membransysteme von E. coli notwendig. In E. coli ist zwar eine Aufnahme von Fettsäuren möglich, aber Triglyceride und Glycerophospholipide müssen in der Zelle bzw. in der Membran synthetisiert werden. Das plsC-Gen ist charakterisiert durch SEQ ID No. 3. Das plsC-Genprodukt (PlsC) ist charakterisiert durch SEQ ID No. 4.
Im Rahmen der vorliegenden Erfindung sind plsC-Homologe Gene, die für ein Protein mit PlsC Aktivität kodieren und die eine Sequenzidentität größer 30% zu SEQ ID No. 3 aufweisen. Besonders bevorzugt ist eine Sequenzidentität größer 70% zu SEQ ID No. 3. Insbesondere bevorzugt handelt es sich um das plsC-Gen.The plsC gene codes for the enzyme 1-acylglycerol-3-phosphate O-acyltransferase (EC: 2.3.1.51). This enzyme transfers a fatty acid from acyl-CoA to an acylglycerol-3-phosphate, releasing a CoA-SH. The resulting diacylglycerol-3-phosphate is used for the synthesis of essential membrane components such as triglycerides and glycerophospholipids. This step is therefore necessary for the production of the membrane systems of E. coli. In E. coli can absorb fatty acids, but triglycerides and glycerophospholipids must be synthesized in the cell or in the membrane. The plsC gene is characterized by SEQ ID no. 3. The plsC gene product (PlsC) is characterized by SEQ ID no. 4th
In the context of the present invention, plsC homologous genes are those which code for a protein with PlsC activity and which have a sequence identity greater than 30% to SEQ ID NO. 3 have. A sequence identity greater than 70% to SEQ ID no. 3. It is particularly preferably the plsC gene.
Besonders bevorzugt weisen PlsC-Homologe eine 1-Acylglycerol-3-Phosphat O-Acyltransferase-Aktivität gemäß EC-Nummer 2.3.1.51 und eine Sequenzidentität größer 70% zu SEQ ID No. 4 auf. Insbesondere bevorzugt handelt es sich um das PlsC Protein.PlsC homologues particularly preferably have a 1-acylglycerol-3-phosphate O-acyltransferase activity according to EC number 2.3.1.51 and a sequence identity greater than 70% with SEQ ID no. 4 on. It is particularly preferably the PlsC protein.
Die PlsC Aktivität in einer Zelle kann entsprechend dem von
Der Grad der DNA-Identität wird durch das Programm "nucleotide blast", zu finden auf der Seite http://blast.ncbi.nlm.nih.gov/, bestimmt, welches auf dem blastn-Algorithmus basiert. Als Algorithmus-Parameter für ein Alignment zweier oder mehrerer Nukleotidsequenzen wurden die voreingestellten Parameter genutzt. Die voreingestellten generellen Parameter sind: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. Die entsprechenden voreingestellten Scoring Parameter sind: Match/Mismatch Scores = 1,-2; Gap Costs = Linear.The degree of DNA identity is determined by the program "nucleotide blast", which can be found on the website http://blast.ncbi.nlm.nih.gov/ , which is based on the blastn algorithm. The preset parameters were used as algorithm parameters for aligning two or more nucleotide sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. The corresponding preset scoring parameters are: Match / Mismatch Scores = 1, -2; Gap Costs = linear.
Für den Vergleich von Proteinsequenzen wird das Programm "protein blast", auf der Seite http://blast.ncbi.nlm.nih.gov/, genutzt. Dieses Programm greift auf den blastp-Algorithmus zurück. Als Algorithmus-Parameter für ein Alignment zweier oder mehrerer Proteinsequenzen wurden die voreingestellten Parameter genutzt. Die voreingestellten generellen Parameter sind: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 3; Automatically adjust parameters for short input sequences = 0. Die voreingestellten Scoring Parameter sind: Matrix = BLOSUM62; Gap Costs = Existence: 11 Extension: 1; Compositional adjustments = Conditional compositional score matrix adjustment.The "protein blast" program on the website http://blast.ncbi.nlm.nih.gov/ is used to compare protein sequences. This program uses the blastp algorithm. The preset parameters were used as algorithm parameters for aligning two or more protein sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect threshold = 10; Word size = 3; Automatically adjust parameters for short input sequences = 0. The preset scoring parameters are: Matrix = BLOSUM62; Gap Costs = Existence: 11 Extension: 1; Compositional adjustments = Conditional compositional score matrix adjustment.
Zur Herstellung eines erfindungsgemäßen Stamms wird in einen geeigneten Mikroorganismenstamm ein temperatursensitives Plasmid eingebracht, welches eine funktionelle Kopie eines nicht fütterbaren Auxotrophie-Gens, das mutiert oder deletiert werden soll, besitzt. Anschließend wird das entsprechende nicht fütterbare Auxotrophie-Gen im Genom des Stammes inaktiviert. Danach wird in diesem Stamm das temperatursensitive Plasmid bei nicht-permissiver Temperatur gegen ein Produktionsplasmid ausgetauscht, wobei das Produktionsplasmid kodierend mindestens ein Enzym zur Produktion einer niedermolekularen Substanz oder mindestens ein rekombinantes Protein auch eine funktionelle Kopie des nicht fütterbaren Auxotrophie-Gens enthält.To produce a strain according to the invention, a temperature-sensitive plasmid is introduced into a suitable microorganism strain which has a functional copy of a non-feedable auxotrophy gene which is to be mutated or deleted. The corresponding non-feedable auxotrophy gene in the genome of the strain is then inactivated. Thereafter, the temperature-sensitive plasmid in this strain is exchanged for a production plasmid at a non-permissive temperature, the production plasmid encoding at least one enzyme for the production of a low molecular weight substance or at least one recombinant protein also containing a functional copy of the non-feedable auxotrophy gene.
Durch dieses Verfahren wird sichergestellt, dass das Produktionsplasmid während eines Fermentationsprozesses zur Herstellung niedermolekularer Verbindungen oder von rekombinanten Proteinen auch in komplexen Medien stabil in der Zelle erhalten bleibt. Somit können erfindungsgemäße Stämme plasmidverlustfrei in Abwesenheit eines zugesetzten selektiven Agens bzw. eines Auxotrophie kompensierenden Additivs kultiviert werden.This process ensures that the production plasmid remains stable in the cell even in complex media during a fermentation process for the production of low molecular weight compounds or recombinant proteins. Thus, strains according to the invention can be cultivated without loss of plasmid in the absence of an added selective agent or an auxotrophy compensating additive.
Als Ausgangsstamm zur Herstellung eines erfindungsgemäßen Stammes ist grundsätzlich jeder Mikroorganismenstamm geeignet, der ein Gen besitzt, dessen Inaktivierung zu einer nicht-fütterbaren Auxotrophie des Stamms führt.In principle, any microorganism strain which has a gene whose inactivation leads to a non-feedable auxotrophy of the strain is suitable as a starting strain for producing a strain according to the invention.
Bevorzugt handelt es sich bei dem Ausgangsstamm zur Erzeugung eines erfindungsgemäßen Stammes um einen Enterobacteriaceen-Stamm, insbesondere bevorzugt um einen Stamm der Spezies Escherichia coli. The starting strain for generating a strain according to the invention is preferably an Enterobacteriaceae strain, particularly preferably a strain of the species E scherichia coli.
Bei den E.coli Stämmen sind solche bevorzugt, die eine "leaky"-Mutation aufweisen. Unter einer "Leaky-Mutation" ist eine Mutation in einem Gen für ein Stukturelement der äußeren Zellmembran oder der Zellwand, ausgewählt aus der Gruppe der omp-Gene, tol-Gene, excD-Gen, excC-Gen, lpp-Gen, pal-Gen, env-Gene und lky-Gene zu verstehen, die dazu führt, dass die Zel-Zellmembran oder der Zellwand, ausgewählt aus der Gruppe der omp-Gene, tol-Gene, excD-Gen, excC-Gen, lpp-Gen, pal-Gen, env-Gene und lky-Gene zu verstehen, die dazu führt, dass die Zellen vermehrt periplasmatische Proteine ins Medium abgeben (
Unter erhöhter Leakiness ist im Sinne der vorliegenden Erfindung zu verstehen, dass nach einer Fermentation der Zellen eine höhere Konzentration von periplasmatischen Proteinen, z. B. der Alkalischen Phosphatase, im Nährmedium vorliegt, als bei einer Fermentation des E. coli-Stammes W3110 (ATCC 27325) unter den gleichen Bedingungen.In the context of the present invention, increased leakiness is to be understood as meaning that, after fermentation of the cells, a higher concentration of periplasmic proteins, e.g. B. the alkaline phosphatase, is present in the nutrient medium than in a fermentation of E. coli strain W3110 (ATCC 27325) under the same conditions.
Verfahren zur Inaktivierung eines Gens in einem Mikroorganismenstamm sind im Stand der Technik bekannt. Sie sind im Folgenden detailliert für die Mutation des plsC-Gens sowie dessen Genprodukte beschrieben. Analog sind diese Verfahren auch für andere Gene, deren Inaktivierung eine nicht fütterbare Auxotrophie eines Mikroorganismenstammes bewirkt, anwendbar. formanten. Bei dem Selektionsmarker handelt es sich beispielsweise um eine Antibiotikaresistenz. Ein bevorzugtes Beispiel für einen temperatursensitiven Replikationsursprung ist "oriR101 & repA101-ts", ein Abkömmling des Replikationsursprunges von Plasmid pSC101 (
Das temperatursensitive Plasmid wird mit einer dem Fachmann bekannten Transformationstechnik, z.B. TSS-, CaCl/RbCl-, Elektroporations-Methode, in die Zelle eingebracht.The temperature-sensitive plasmid is introduced into the cell using a transformation technique known to the person skilled in the art, e.g. TSS, CaCl / RbCl, electroporation method.
Auf Zellen, die das temperatursensitive Plasmid beinhalten, wird mittels des Selektionsmarkers, der auf dem temperatursensitiven Plasmid vorhanden ist, selektiert, während die Zellen bei für das Plasmid permissiver Temperatur inkubiert werden.Cells which contain the temperature-sensitive plasmid are selected by means of the selection marker which is present on the temperature-sensitive plasmid, while the cells are incubated at a temperature permissive for the plasmid.
Ein derartiges temperatursensitives Plasmid lässt sich gegen das Produktionsplasmid austauschen, indem der Mikroorganismenstamm nach der Transformation mit dem Produktionsplasmid bei einer für das temperatursensitive Plasmid nicht-permissiven Temperatur kultiviert wird. Ein bevorzugter nicht-permissiver Temperaturbereich ist 37-45°C, besonders bevorzugt 39-43°C.Such a temperature-sensitive plasmid can be exchanged for the production plasmid by cultivating the microorganism strain after the transformation with the production plasmid at a temperature which is non-permissive for the temperature-sensitive plasmid. A preferred non-permissive temperature range is 37-45 ° C, particularly preferably 39-43 ° C.
Das Produktionsplasmid wird mit einer dem Fachmann bekannten Transformationstechnik, z.B. TSS-, CaC1/RbCl-, Elektroporations-Methode, in die Zelle eingebracht.The production plasmid is introduced into the cell using a transformation technique known to the person skilled in the art, e.g. TSS, CaC1 / RbCl, electroporation method.
Die Kultivierung der Transformanten kann sowohl auf AgarPlatten wie in Flüssigkultur erfolgen. In einer bevorzugten Ausführungsform wird der Mikroorganismenstamm unmittelbar nach der Transformation mit dem Produktionsplasmid für 30-90 min, bevorzugt 60 min, einem Temperaturschock bei 47-55°C, bevorzugt bei 52°C, ausgesetzt. Anschließend erfolgt die weitere Inkubation bei der oben genannten nicht permissiven Temperatur. Dadurch wird in einem Schritt das temperatursensitive Plasmid gegen das Produktionsplasmid ausgetauscht und ein erfindungsgemäßer Produktionsstamm zur antibiotikafreien Produktion von niedermolekularen Substanzen oder rekombinanten Proteinen generiert.The transformants can be cultivated on agar plates or in liquid culture. In a preferred embodiment, the microorganism strain is exposed to a temperature shock at 47-55 ° C., preferably at 52 ° C., for 30-90 min, preferably 60 min, immediately after the transformation with the production plasmid. Further incubation then takes place at the non-permissive temperature mentioned above. As a result, the temperature-sensitive plasmid is exchanged for the production plasmid and a plasmid according to the invention in one step Production strain generated for the antibiotic-free production of low molecular weight substances or recombinant proteins.
Als Produktionsplasmid wird beispielsweise einer der folgenden bekannten Expressionsvektoren verwendet: pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3 oder pET.One of the following known expression vectors is used, for example, as the production plasmid: pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3 or pET.
Das Produktionsplasmid enthält neben der funktionellen Kopie des nicht fütterbaren Auxotrophie-Gens ein oder mehrere Zielgene sowie die zur Expression dieser Zielgene notwendigen Expressionssignale, wie z.B. Promotor-, Operator-, und Terminatorsequenzen. Die Zielgene kodieren für mindestens ein Enzym zur Produktion einer niedermolekularen Substanz oder mindestens ein rekombinantes Protein.
Bei den niedermolekularen Substanzen handelt es sich um Grundbausteine (Basen, Aminosäuren, Fettsäuren etc.) sowie Sekundärmetabolite (Vitamine, Antioxidantien etc.) die der Organismus synthetisieren kann. Bevorzugt sind Aminosäuren, besonders bevorzugt die Aminosäure L-Cystein und davon abgeleitete Verbindungen.In addition to the functional copy of the non-feedable auxotrophy gene, the production plasmid contains one or more target genes and the expression signals necessary for expressing these target genes, such as promoter, operator and terminator sequences. The target genes code for at least one enzyme for the production of a low molecular weight substance or at least one recombinant protein.
The low molecular weight substances are basic building blocks (bases, amino acids, fatty acids etc.) as well as secondary metabolites (vitamins, antioxidants etc.) that the organism can synthesize. Amino acids are preferred, the amino acid L-cysteine and compounds derived therefrom are particularly preferred.
Bei den rekombinanten Proteinen handelt es sich vorzugsweise um heterologe Proteine.The recombinant proteins are preferably heterologous proteins.
Unter einem heterologen Protein ist ein Protein zu verstehen, das nicht zum Proteom, d.h. der gesamten natürlichen Protein-Ausstattung, des Wirtsorganismus gehört.A heterologous protein is understood to be a protein that does not belong to the proteome, i.e. the entire natural protein structure of the host organism.
Bevorzugt handelt es sich bei dem heterologen Protein um ein eukaryotisches Protein, besonders bevorzugt um ein Protein, welches eine oder mehrere Disulfid-Brücken enthält oder welches in seiner funktionellen Form als Di- oder Multimer vorliegt, d.h. dass das Protein eine Quartärstruktur besitzt und aus mehreren identischen (homologen) oder nicht-identischen (heterologen) Untereinheiten aufgebaut ist.The heterologous protein is preferably a eukaryotic protein, particularly preferably a protein which contains one or more disulfide bridges or which is present in its functional form as a dimer or multimer, ie the protein has a quaternary structure and consists of several identical (homologous) or non-identical (heterologous) subunits.
Eine bevorzugte Klasse von Proteinen, die aus mehreren Proteinuntereinheiten besteht, sind Antikörper oder Fragmente von Antikörpern. Besonders bevorzugt sind funktionelle Fab-Antikörperfragmente.A preferred class of proteins that consists of several protein subunits are antibodies or fragments of antibodies. Functional Fab antibody fragments are particularly preferred.
Im Folgenden sind Gegenstände, die sich auf das pyrH-Gen beziehen, nicht Teil der Erfindung und dienen nur der Veranschaulichung. Im Folgenden wird die Herstellung eines Mikroorganismenstammes beschrieben, bei dem das nicht fütterbare Auxotrophie-Gen pyrH mutiert ist.In the following, items relating to the pyrH gene do not form part of the invention and are for illustrative purposes only. The following describes the production of a microorganism strain in which the non-feedable auxotrophy gene pyrH is mutated.
Als Ausgangsstämme für die Herstellung eines Mikroorganismenstammes mit einer genomischen pyrH Inaktvierung ist grundsätzlich jeder Wirtsorganismus geeignet, der ein Gen für die UMP-Kinase PyrH besitzt.In principle, any host organism that has a gene for the UMP kinase PyrH is suitable as starting strains for the production of a microorganism strain with genomic pyrH inactivation.
Verfahren zur Inaktivierung des pyrH-Gens in einem Mikroorganismenstamm sind im Stand der Technik bekannt. Das pyrH-Gen kann beispielsweise dadurch inaktiviert werden, dass eine Mutation (Substitution, Insertion oder Deletion einzelner oder mehrerer Nukleotide) in den Leserahmen des pyrH-Gens eingebracht wird, welche dazu führt, dass die spezifische Aktivität von PyrH inaktiviert wird. Dem Fachmann sind Verfahren zur Erzeugung solcher pyrH-Allele bekannt. So kann beispielsweise mittels des in
Darüber hinaus kann das pyrH-Gen einer Zelle auch dadurch inaktiviert werden, dass mindestens ein für die Expressionsregulation notwendiges Element (z.B. Promotor, Enhancer, Ribosomen-Bindestelle) durch Substitution, Insertion oder Deletion einzelner oder mehrerer Nukleotide mutiert wird. Eine Inaktivierung im Sinne der Erfindung liegt vor, wenn die Wachstumsrate der Zellen in einer Fermentation durch die Inaktivierung des Gens im Vergleich zu den Zellen vor der Mutation auf ≤ 10% erniedrigt ist, bevorzugt wenn kein Wachstum mehr stattfindet. Besonders bevorzugt führt die Mutation zum Tod der Mikroorganismenzelle.In addition, the pyrH gene of a cell can also be inactivated by using at least one element necessary for expression regulation (e.g. promoter, enhancer, ribosome binding site) is mutated by substitution, insertion or deletion of one or more nucleotides. Inactivation within the meaning of the invention is when the growth rate of the cells in a fermentation is reduced to 10% compared to the cells before the mutation due to the inactivation of the gene, preferably when there is no longer any growth. The mutation particularly preferably leads to the death of the microorganism cell.
Da die Inaktivierung des pyrH-Gens zu einer nicht-fütterbaren Auxotrophie führt, muss eine funktionelle Kopie des pyrH-Gens bereits vor der chromosomalen Inaktivierung in der Zelle vorhanden sein. Dies kann durch das transiente Einbringen eines temperatursensitiven Plasmids, das eine funktionelle Kopie des pyrH-Gens enthält, erreicht werden.Since the inactivation of the pyrH gene leads to a non-feedable auxotrophy, a functional copy of the pyrH gene must already be present in the cell before the chromosomal inactivation. This can be achieved by the transient introduction of a temperature-sensitive plasmid containing a functional copy of the pyrH gene.
In solchen Zellen, die das temperatursensitive Plasmid enthalten, kann nun durch die genannten Methoden das pyrH-Gen im Genom inaktiviert werden.In those cells that contain the temperature-sensitive plasmid, the pyrH gene in the genome can now be inactivated using the methods mentioned.
In einem weiteren Schritt wird dieses temperatursensitive Plasmid gegen das Produktionsplasmid, das auch eine funktionelle Kopie des pyrH-Gens enthält, ausgetauscht. Dies geschieht vorzugsweise in der bereits beschriebenen Art und Weise.In a further step, this temperature-sensitive plasmid is exchanged for the production plasmid, which also contains a functional copy of the pyrH gene. This is preferably done in the manner already described.
Analog lassen sich erfindungsgemäße Mikroorganismenstämme mit einem inaktivierten plsC-Gen herstellen.Microorganism strains according to the invention with an inactivated plsC gene can be produced analogously.
Bei der Herstellung von rekombinanten Proteinen unterscheidet man zwischen einer cytoplasmatischen und einer sekretorischen Produktion. Während bei der cytoplasmatischen Produktion das Zielprotein im Cytoplasma der Zelle angehäuft wird, wird bei der sekretorischen Produktion das Zielprotein in das Periplasma bzw. in das Kulturmedium transloziert. Im Rahmen der Erfindung wird die sekretorische Produktion bevorzugt. Insbesondere bevorzugt ist die sekretorische Produktion des Zielproteins in das Kulturmedium.In the production of recombinant proteins, a distinction is made between cytoplasmic and secretory production. While in cytoplasmic production the target protein is accumulated in the cytoplasm of the cell, in secretory production the target protein is translocated into the periplasm or into the culture medium. Secretory production is preferred within the scope of the invention. The secretory production of the target protein in the culture medium is particularly preferred.
Für die sekretorische Produktion von Proteinen, d.h. für die Translokation des Proteins aus dem Cytoplasma in das Periplasma bzw. das Kulturmedium, ist es notwendig, das 5'-Ende des Gens des zu produzierenden Proteins in frame mit dem 3'-Ende einer Signalsequenz für den Protein-Export zu verknüpfen. Dazu sind prinzipiell die Gene aller Signalsequenzen geeignet, die in E. coli zu einer Translokation des Zielproteins in das Periplasma führen. In E. coli sind drei Haupttranslokationswege bekannt: der SEC-, TAT- und SRP-Weg. Bevorzugt sind solche Signalsequenzen, die eine Translokation über den SEC-Apparat ermöglichen. Verschiedene derartige Signalsequenzen sind im Stand der Technik beschrieben, so z.B. die Signalsequenzen der folgenden Gene: phoA, ompA, pelB, ompF, ompT, lamB, malE, dsbA, Staphylococcal protein A, StII und andere (
Die Kultivierung (Fermentation) der Zellen, die ein Produktionsplasmid enthalten, erfolgt nach üblichen, dem Fachmann bekannten Fermentationsverfahren in einem Bioreaktor (Fermenter) ohne Zugabe von Antibiotika.For the secretory production of proteins, ie for the translocation of the protein from the cytoplasm into the periplasm or the culture medium, it is necessary to place the 5 'end of the gene of the protein to be produced in frame with the 3' end of a signal sequence for to link the protein export. In principle, the genes of all signal sequences that lead to translocation of the target protein into the periplasm in E. coli are suitable for this. Three main translocation pathways are known in E. coli : the SEC, TAT and SRP pathways. Signal sequences which enable translocation via the SEC apparatus are preferred. Various such signal sequences are described in the prior art, for example the signal sequences of the following genes: phoA, ompA, pelB, ompF, ompT, lamB, malE, dsbA, staphylococcal protein A, StII and others (
The cultivation (fermentation) of the cells which contain a production plasmid takes place according to conventional fermentation processes known to the person skilled in the art in a bioreactor (fermenter) without the addition of antibiotics.
Die Erfindung betrifft auch ein Verfahren zur Herstellung von niedermolekularen Substanzen oder rekombinanten Proteinen mittels eines Mikroorganismenstamms, das dadurch gekennzeichnet ist, dass ein erfindungsgemäßer Mikroorganismenstamm eingesetzt wird und ein antibiotikafreies Fermentationsmedium verwendet wird.The invention also relates to a method for producing low molecular weight substances or recombinant proteins by means of a microorganism strain, which is characterized in that a microorganism strain according to the invention is used and an antibiotic-free fermentation medium is used.
Die Fermentation findet in einem üblichen Bioreaktor, beispielsweise einem Rührkessel, einem Blasensäulen-Fermenter oder einem Airlift-Fermenter statt. Bevorzugt ist ein Rührkessel-Fermenter im technischem Maßstab und damit mit einer Größe von >100 1.The fermentation takes place in a conventional bioreactor, for example a stirred tank, a bubble column fermenter or an airlift fermenter. A stirred tank fermenter on an industrial scale and thus with a size of> 100 1 is preferred.
Bei der Fermentation werden die Zellen des erfindungsgemäßen Stammes in einem Flüssigmedium kultiviert, wobei verschiedene Parameter wie z.B. die Nährstoffzufuhr, der Sauerstoff-Partialdruck, der pH-Wert und die Temperatur der Kultur laufend kontrolliert und genau gesteuert werden. Der Zeitraum der Kultivierung beträgt vorzugsweise 16-150 h, besonders bevorzugt 24-72 h.During fermentation, the cells of the strain according to the invention are cultivated in a liquid medium, with various parameters such as the nutrient supply, the oxygen partial pressure, the pH value and the temperature of the culture being continuously and precisely controlled. The cultivation period is preferably 16-150 hours, particularly preferably 24-72 hours.
Als Fermentationsmedien kommen alle gängigen, dem Fachmann bekannten Medien für die Kultivierung von Mikroorganismen in Frage. Diese Fermentationsmedien sind jedoch frei von einem Antibiotikum.All common media known to the person skilled in the art for the cultivation of microorganisms can be used as fermentation media. However, these fermentation media are free from an antibiotic.
Es können Komplexmedien oder Minimalsalzmedien, denen ein definierter Anteil von Komplex-Komponenten wie z.B. Pepton, Trypton, Hefeextrakt, Melasse oder Corn Steep Liquor zugesetzt wird, verwendet werden. Bevorzugt wird ein Medium mit Komplexmedienkomponenten.Complex media or minimal salt media to which a defined proportion of complex components such as peptone, tryptone, yeast extract, molasses or corn steep liquor is added can be used. A medium with complex media components is preferred.
Als primäre Kohlenstoffquelle für die Fermentation können alle von den Zellen verwertbaren Zucker, Zuckeralkohole oder organische Säuren bzw. deren Salze verwendet werden. Dabei werden bevorzugt Glucose, Laktose oder Glycerin eingesetzt. Besonders bevorzugt sind Glucose und Laktose. Auch ist eine kombinierte Fütterung mehrerer verschiedener Kohlenstoffquellen möglich. Die Kohlenstoffquelle kann dabei zu Beginn der Fermentation vollständig im Fermentationsmedium vorgelegt werden oder es wird nichts oder nur ein Teil der Kohlenstoffquelle zu Beginn vorgelegt und die Kohlenstoffquelle wird über den Verlauf der Fermentation zugefüttert. Besonders bevorzugt ist dabei eine Ausführungsform, bei der ein Teil der Kohlenstoffquelle vorgelegt wird und ein Teil gefüttert wird. Besonders bevorzugt wird die Kohlenstoffquelle in einer Konzentration von 10-30 g/l vorgelegt, die Fütterung gestartet, wenn die Konzentration auf kleiner 5 g/l abgefallen ist und so gestaltet, dass die Konzentration unter 5 g/l gehalten wird.All sugars, sugar alcohols or organic acids or their salts which can be used by the cells can be used as the primary carbon source for the fermentation. Glucose, lactose or glycerine are preferably used. Glucose and lactose are particularly preferred. Combined feeding of several different carbon sources is also possible. The carbon source can be placed completely in the fermentation medium at the beginning of the fermentation or nothing or only a part of the carbon source is placed at the beginning and the carbon source is fed in over the course of the fermentation. An embodiment is particularly preferred in which part of the carbon source is presented and part is fed. The carbon source is particularly preferably provided in a concentration of 10-30 g / l, feeding is started when the concentration has fallen to less than 5 g / l and is designed so that the concentration is kept below 5 g / l.
Der Sauerstoff-Partialdruck (pO2) in der Kultur beträgt vorzugsweise zwischen 10 und 70 % Sättigung. Bevorzugt wird ein pO2 zwischen 20 und 60 %, besonders bevorzugt liegt der pO2 zwischen 45 und 55 % Sättigung.The oxygen partial pressure (pO 2 ) in the culture is preferably between 10 and 70% saturation. A pO 2 between 20 and 60% is preferred, the pO 2 is particularly preferably between 45 and 55% saturation.
Der pH-Wert der Kultur liegt vorzugsweise zwischen pH 6 und pH 8. Bevorzugt wird ein pH-Wert zwischen 6,5 und 7,5, besonders bevorzugt liegt der pH-Wert der Kultur zwischen 6,8 und 7,2.The pH value of the culture is preferably between pH 6 and pH 8. A pH value between 6.5 and 7.5 is preferred, and the pH value of the culture is particularly preferably between 6.8 and 7.2.
Die Temperatur der Kultur liegt zwischen 15 und 45 °C. Bevorzugt ist ein Temperaturbereich zwischen 18 und 40 °C, besonders bevorzugt ist ein Temperaturbereich zwischen 25 und 35 °C, ganz besonders bevorzugt sind 30 °C.The temperature of the culture is between 15 and 45 ° C. A temperature range between 18 and 40 ° C. is preferred, a temperature range between 25 and 35 ° C. is particularly preferred, and 30 ° C. is very particularly preferred.
Bei einer bevorzugten Ausrichtung handelt es sich bei der Fermentation um eine Hochzelldichte-Fermentation. Unter einer Hochzelldichte-Fermentation ist eine Fermentation zu verstehen, in deren Verlauf Zelltrockengewichte von mehr als 50 g/l erreicht werden. Besonders bevorzugt werden Zelltrockengewichte von mehr als 70 g/l.
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Fig. 1 zeigt eine Restriktions- und Funktionskarte von Plasmid pKD46 aus Bsp. 2. -
Fig. 2 zeigt eine Restriktions- und Funktionskarte des Plasmids pAF-ts-pyrH hergestellt in Bsp. 2. -
Fig. 3 zeigt eine Restriktions- und Funktionskarte des Plasmids pAF-ts-plsC hergestellt in Bsp. 2. -
Fig. 4 zeigt eine Restriktions- und Funktionskarte das Plasmids pMT1 verwendet in Bsp. 5. -
Fig. 5 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pcysEX-GAPDH-ORF306_tetR hergestellt in Bsp. 5. -
Fig. 6 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pCGT_tetR hergestellt in Bsp. 6. -
Fig. 7 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pFab-anti-Lysozym_tetR hergestellt in Bsp. 7. -
Fig. 8 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pcysEX-GAPDH-ORF306_pyrH1_tetR hergestellt in Bsp. 8. -
Fig. 9 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pCGT_pyrH1_tetR hergestellt in Bsp. 8. -
Fig. 10 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pFab-anti-Lysozyme_pyrH1_tetR hergestellt in Bsp. 8. -
Fig. 11 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pcysEX-GAPDH-ORF306_plsC1_tetR hergestellt in Bsp. 8. -
Fig. 12 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pCGT_plsC1_tetR hergestellt in Bsp. 8. -
Fig. 13 zeigt eine Restriktions- und Funktionskarte des Expressionsplasmids pFab-anti-Lysozyme_plsC1_tetR hergestellt in Bsp. 8. -
Fig. 14 zeigt eine Restriktions- und Funktionskarte des erfindungsgemäßen Produktionsplasmids pcysEX-GAPDH-ORF306_pyrH hergestellt in Bsp. 9. -
Fig. 15 zeigt eine Restriktions- und Funktionskarte des erfindungsgemäßen Produktionsplasmids pCGT_pyrH hergestellt in Bsp. 9. -
Fig. 16 zeigt eine Restriktions- und Funktionskarte des erfindungsgemäßen Produktionsplasmids pFab-anti-Lysozym_pyrH hergestellt in Bsp. 9. -
Fig. 17 zeigt eine Restriktions- und Funktionskarte des erfindungsgemäßen Produktionsplasmids pcysEX-GAPDH-ORF306_plsC hergestellt in Bsp. 9. -
Fig. 18 zeigt eine Restriktions- und Funktionskarte des erfindungsgemäßen Produktionsplasmids pCGT_plsC hergestellt in Bsp. 9. -
Fig. 19 zeigt eine Restriktions- und Funktionskarte des erfindungsgemäßen Produktionsplasmids pFab-anti-Lysozym_plsC hergestellt in Bsp. 9.
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Fig. 1 shows a restriction and function map of plasmid pKD46 from Ex. -
Fig. 2 shows a restriction and function map of the plasmid pAF-ts-pyrH produced in Ex. -
Fig. 3 shows a restriction and function map of the plasmid pAF-ts-plsC produced in Ex. -
Fig. 4 shows a restriction and function map of the plasmid pMT1 used in Ex. -
Fig. 5 shows a restriction and function map of the expression plasmid pcysEX-GAPDH-ORF306_tetR produced in Ex. 5. -
Fig. 6 shows a restriction and function map of the expression plasmid pCGT_tetR produced in Ex. 6. -
Fig. 7 shows a restriction and function map of the expression plasmid pFab-anti-Lysozyme_tetR produced in Ex. -
Fig. 8 shows a restriction and function map of the expression plasmid pcysEX-GAPDH-ORF306_pyrH1_tetR produced in Ex. 8. -
Fig. 9 shows a restriction and function map of the expression plasmid pCGT_pyrH1_tetR produced in Ex. 8. -
Fig. 10 shows a restriction and function map of the expression plasmid pFab-anti-Lysozyme_pyrH1_tetR produced in Ex. 8. -
Fig. 11 shows a restriction and function map of the expression plasmid pcysEX-GAPDH-ORF306_plsC1_tetR produced in Ex. 8. -
Fig. 12 shows a restriction and function map of the expression plasmid pCGT_plsC1_tetR produced in Ex. 8. -
Fig. 13 shows a restriction and function map of the expression plasmid pFab-anti-Lysozyme_plsC1_tetR produced in Ex. 8. -
Fig. 14 shows a restriction and function map of the production plasmid pcysEX-GAPDH-ORF306_pyrH produced in Ex. 9 according to the invention. -
Fig. 15 shows a restriction and function map of the production plasmid pCGT_pyrH according to the invention produced in Ex. 9. -
Fig. 16 shows a restriction and function map of the production plasmid pFab-anti-Lysozyme_pyrH produced in Ex. 9 according to the invention. -
Fig. 17 shows a restriction and function map of the production plasmid pcysEX-GAPDH-ORF306_plsC according to the invention produced in Ex. 9. -
Fig. 18 FIG. 9 shows a restriction and function map of the production plasmid pCGT_plsC according to the invention produced in Ex. 9. -
Fig. 19 shows a restriction and function map of the production plasmid pFab-anti-Lysozyme_plsC according to the invention produced in Ex. 9.
Die folgenden Beispiele dienen der weiteren Erläuterung der Erfindung. Sämtliche eingesetzten molekularbiologischen und mikrobiologischen Verfahren wie Polymerase-Ketten-Reaktion (PCR), Gensynthese, Isolierung und Reinigung von DNA, Modifikation von DNA durch Restriktionsenzyme, Klenow-Fragment und Ligase, Transformation, P1-Transduktion etc., wurden in der dem Fachmann bekannten, in der Literatur beschriebenen oder von den jeweiligen Herstellern empfohlenen Art und Weise durchgeführt. Die folgenden Beispiele, sofern sie sich auf das pyrH-Gen beziehen, entsprechen nicht der Erfindung und dienen nur der Veranschaulichung.The following examples serve to further illustrate the invention. All molecular biological and microbiological methods used, such as polymerase chain reaction (PCR), gene synthesis, isolation and purification of DNA, modification of DNA by restriction enzymes, Klenow fragment and ligase, transformation, P1 transduction, etc., were included in the carried out in a manner known to the person skilled in the art, described in the literature or recommended by the respective manufacturers. The following examples, insofar as they relate to the pyrH gene, do not correspond to the invention and are provided for illustration only.
Ein etwa 1,0 kb großes DNA-Fragment, welches für das pyrH-Gen inklusive nativer Promotoregion kodiert, wurde mit den Primern pyrH-NcoI-fw (SEQ ID No. 5) und pyrH-NcoI-rev (SEQ ID No. 6) amplifiziert. Als Matrize für die PCR-Reaktion diente chromosomale DNA des E. coli Stammes W3110 (ATCC 27325).An approximately 1.0 kb DNA fragment which codes for the pyrH gene including the native promoter region was prepared with the primers pyrH-NcoI-fw (SEQ ID No. 5) and pyrH-NcoI-rev (SEQ ID No. 6 ) amplified. Chromosomal DNA of E served as a template for the PCR reaction. coli strain W3110 (ATCC 27325).
Das etwa 1,0 kb große PCR-Fragment wurde über eine Agarose-Gelelektrophorese gereinigt und mit dem "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, D) nach Angaben des Herstellers aus dem Agarosegel isoliert. Anschließend wurde das gereinigte PCR-Fragment mit dem Restriktionsenzym NcoI verdaut und bei -20°C gelagert. Analog wurde ein etwa 1,0 kb großes DNA-Fragment, welches für das plsC-Gen inklusive nativer Promotorregion kodiert, amplifiziert, gereinigt, verdaut und gelagert. Für die Amplifikation wurden die Primer plsC-NcoI-fw (SEQ ID No. 7) und plsC-NcoI-rev (SEQ ID No. 8) verwendet.The approximately 1.0 kb PCR fragment was purified by agarose gel electrophoresis and isolated from the agarose gel using the "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, D) according to the manufacturer's instructions. The purified PCR fragment was then digested with the restriction enzyme NcoI and stored at -20.degree. Similarly, an approximately 1.0 kb DNA fragment, which codes for the plsC gene including the native promoter region, was amplified, purified, digested and stored. The primers plsC-NcoI-fw (SEQ ID No. 7) and plsC-NcoI-rev (SEQ ID No. 8) were used for the amplification.
Als Ausgangsplasmid für die Konstruktion der Plasmide pAF-ts-pyrH und pAF-ts-plsC mit temperatur-sensitivem Replikationsursprung wurde das Plasmid pKD46 verwendet (
Die in Beispiel 2 beschriebenen Plasmide pAF-ts-pyrH und pAF-ts-plsC mit temperatur-sensitivem Replikationsursprung wurden mit der dem Fachmann bekannten CaCl2-Methode in die beiden E. coli Stämme W3110 (ATCC 27325) und W31101pp3 (beschrieben in
Das Gen pyrH, welches in E. coli für das Enzym Uridylatkinase (PyrH) kodiert, wurde nach der von Datsenko und Wanner entwickelten "λ-Red-Methode" in den E. coli Stämmen W3110/pAF-ts-pyrH und W31101pp3/pAF-ts-pyrH deletiert (
Das amplifizierte PCR-Produkt wurde mittels Elektroporation in die E. coli Stämme W3110/pAFts-pyrH und W3110lpp3/pAF-ts-pyrH (siehe Beispiel 3) eingebracht. Die Selektion auf Zellen mit chromosomaler Integration des Kanamycin-Resistenz-Markergens (kanR) erfolgte auf LB-Agarplatten, die 50 mg/l Kanamycin und 100 mg/l Ampicillin enthielten. Die Entfernung des chromosomal eingebrachten Kanamycin-Resistenz-Markergens (kanR) erfolgte mit dem Enzym FLP-Rekombinase, welches auf dem Plasmid pCP20 (CGSC No. 7629) kodiert ist. Die Selektion auf pCP20-enthaltende Zellen erfolgte auf LB-Agarplatten, die 100 mg/l Ampicillin (Selektion auf pAF-ts-pyrH) und 34 mg/l Chloramphenicol (Selektion auf pCP20) enthielten. Aufgrund eines temperatursensitiven Replikationsursprunges (ori) kann das Plasmid pCP20 nach erfolgter Transformation durch Kultivierung der E. coli Zellen bei nicht permissiver, d.h. erhöhter Temperatur, z.B. bei 42°C, wieder entfernt werden.The amplified PCR product was introduced into the E. coli strains W3110 / pAFts-pyrH and W3110lpp3 / pAF-ts-pyrH (see Example 3) by means of electroporation. The selection for cells with chromosomal integration of the kanamycin resistance marker gene ( kanR ) was carried out on LB agar plates containing 50 mg / l kanamycin and 100 mg / l ampicillin. The chromosomally introduced kanamycin resistance marker gene ( kanR ) was removed with the enzyme FLP recombinase, which is encoded on the plasmid pCP20 (CGSC No. 7629). The selection for pCP20-containing cells took place on LB agar plates which contained 100 mg / l ampicillin (selection for pAF-ts-pyrH) and 34 mg / l chloramphenicol (selection for pCP20). Due to a temperature-sensitive origin of replication (ori), the plasmid pCP20 can be removed again after transformation by culturing the E. coli cells at a non-permissive, ie elevated temperature, for example at 42 ° C.
Eine erste Selektion auf Verlust des temperatursensitiven Plasmids pCP20 bei gleichzeitigem Erhalt des temperatur-sensitiven Plasmids pAF-ts-pyrH, erfolgte auf LB-Agarplatten, die 100 mg/l Ampicillin enthielten (Selektion auf pAF-ts-pyrH). Im weiteren Verlauf wurden die vorselektierten Ampicillin-resistenten Bakterien-Klone auf Kanamycin-Sensitivität, d.h. den Verlust des chromosomal eingebrachten Kanamycin-Markergens, sowie auf Chloramphenicol-Sensitivität, d.h. den Verlust des temperatursensitiven Plasmids pCP20 überprüft.A first selection for loss of the temperature-sensitive plasmid pCP20 with simultaneous receipt of the temperature-sensitive plasmid pAF-ts-pyrH was carried out on LB agar plates which contained 100 mg / l ampicillin (selection for pAF-ts-pyrH). Subsequently, the preselected ampicillin-resistant bacterial clones were checked for kanamycin sensitivity, i.e. the loss of the chromosomally introduced kanamycin marker gene, as well as for chloramphenicol sensitivity, i.e. the loss of the temperature-sensitive plasmid pCP20.
Nur Kanamycin-, sowie Chloramphenicol-sensitive aber Ampicillin-resistente Klone wurden abschließend mit den Primern pyrH-check-for (SEQ ID No. 11) und pyrH-check-rev (SEQ ID No. 12) auf die chromosomale Deletion des pyrH-Gens hin überprüft. Als Template für die Überprüfung der chromosomalen pyrH-Deletion mittels PCR diente chromosomale DNA der selektierten Ampicillin-resistenten, Chloramphenicol- und Kanamycin-sensitiven Klone.Only kanamycin- and chloramphenicol-sensitive but ampicillin-resistant clones were finally checked for the chromosomal deletion of the pyrH - with the primers pyrH-check-for (SEQ ID No. 11) and pyrH -check-rev (SEQ ID No. 12) Gens checked. Chromosomal DNA of the selected ampicillin-resistant, chloramphenicol- and kanamycin-sensitive clones served as a template for checking the chromosomal pyrH deletion by means of PCR.
Die auf diese Art und Weise generierten und überprüften Ampicillin-resistenten E. coli-Stämme mit chromosomaler pyrH-Deletion und Plasmid-kodierter pyrH-Expression tragen die Bezeichnungen W3110ΔpyrH/pAF-ts-pyrH und W3110lpp3ΔpyrH/pAF-ts-pyrH.The ampicillin-resistant E. coli strains with chromosomal pyrH deletion and plasmid-encoded pyrH expression have the designations W3110ΔpyrH / pAF-ts-pyrH and W3110lpp3ΔpyrH / pAF-ts-pyrH.
Analog zum pyrH-Gen wurde das Gen plsC, welches in E. coli für das Enzym 1-Acylglycerol-3-Phosphat O-Acyltransferase (PlsC) kodiert, in den E. coli Stämmen W3110/pAF-ts-plsC und W31101pp3/pAF-ts-plsC deletiert (
Das amplifizierte PCR-Produkt wurde mittels Elektroporation in die E. coli Stämme W3110/pAFts-plsC und W3110lpp3/pAF-ts-plsC (siehe Beispiel 3) eingebracht. Die Entfernung des chromosomal eingebrachten Kanamycin-Resistenz-Markergens (kanR) erfolgte wieder mit dem Enzym FLP-Rekombinase (kodiert auf Plasmid pCP20).Die Selektion auf pCP20-enthaltene Zellen erfolgte auch hier auf LB-Agarplatten, die 100 mg/l Ampicillin (Selektion auf pAF-ts-plsC) und 34 mg/l Chloramphenicol (Selektion auf pCP20) enthielten. Eine erste Selektion auf Verlust des temperatursensitiven Plasmids pCP20 bei gleichzeitigem Erhalt des temperatur-sensitiven Plasmids pAF-ts-plsC, erfolgte auf LB-Agarplatten, die 100 mg/l Ampicillin enthielten (Selektion auf pAF-ts-plsC). Im weiteren Verlauf wurden die vorselektierten Ampicillin-resistenten Bakterien-Klone auf Kanamycin-Sensitivität, d.h. den Verlust des chromosomal eingebrachten Kanamycin-Markergens, sowie auf Chloramphenicol-Sensitivität, d.h. den Verlust des temperatursensitiven Plasmids pCP20 überprüft. Diese Klone wurden abschließend mit den Primern plsC-check-for (SEQ ID No. 15) und plsC-check-rev (SEQ ID No. 16) auf die chromosomale Deletion des plsC-Gens hin überprüft. Als Template für die Überprüfung der chromosomalen plsC-Deletion mittels PCR diente chromosomale DNA der selektierten Ampicillin-resistenten, Chloramphenicol- und Kanamycin-sensitiven Klone.The amplified PCR product was introduced into the E. coli strains W3110 / pAFts-plsC and W3110lpp3 / pAF-ts-plsC (see Example 3) by means of electroporation. The chromosomally introduced kanamycin resistance marker gene ( kanR ) was removed again with the enzyme FLP recombinase (encoded on plasmid pCP20). The selection for pCP20-containing cells was also carried out here on LB agar plates containing 100 mg / l ampicillin ( Selection for pAF-ts-plsC) and 34 mg / l chloramphenicol (selection for pCP20). A first selection for loss of the temperature-sensitive plasmid pCP20 with simultaneous receipt of the temperature-sensitive plasmid pAF-ts-plsC was made on LB agar plates containing 100 mg / l ampicillin (selection for pAF-ts-plsC). Subsequently, the preselected ampicillin-resistant bacterial clones were checked for kanamycin sensitivity, ie the loss of the chromosomally introduced kanamycin marker gene, and for chloramphenicol sensitivity, ie the loss of the temperature-sensitive plasmid pCP20. These clones were then with the primers plsC-check-for (SEQ ID No. 15) and plsC-check-rev (SEQ ID No. 16) on the chromosomal deletion of the plsC gene checked. Chromosomal DNA of the selected ampicillin-resistant, chloramphenicol- and kanamycin-sensitive clones served as a template for checking the chromosomal plsC deletion by means of PCR.
Die auf diese Art und Weise generierten und überprüften Ampicillin-resistenten E. coli-Stämme mit chromosomaler plsC-Deletion und Plasmid-kodierter plsC-Expression tragen die Bezeichnungen W3110ΔplsC/pAF-ts-plsC und W3110lpp3ΔplsC/pAF-ts-plsC.The ampicillin-resistant E. coli strains with chromosomal plsC deletion and plasmid-encoded plsC expression have the designations W3110ΔplsC / pAF-ts-plsC and W3110lpp3ΔplsC / pAF-ts-plsC.
Als Ausgangsplasmide für die Klonierung und Expression der Gene cysEX (kodiert für feedbackresistente Variante der Serin Acyltransferase; CysE) und orf306 (kodiert für O-Acetylserin / Cystein-Exporter; EamA) dienten das Basisplasmid pMT1 sowie das in
pMT1 enthält neben dem Tetracyclin-Resistenzgen (tetR) noch den tac-Promotor, der durch das LacIq-Genprodukt, dessen Gen ebenfalls auf dem Plasmid vorliegt, reprimiert wird und der durch einen Induktor wie z.B. D-Lactose oder Isopropyl-β-D-thiogalactopyranosid (IPTG) angeschaltet werden kann.In addition to the tetracycline resistance gene (tetR), pMT1 also contains the tac promoter, which is repressed by the LacIq gene product, the gene of which is also present on the plasmid, and which is produced by an inducer such as D-lactose or isopropyl-β-D- thiogalactopyranoside (IPTG) can be switched on.
Eine Restriktions- und Funktionskarte von Plasmid pMT1 ist in
Für die Generierung eines neuen Produktionsplasmides für die Produktion von Cystein, auf Basis von pMT1, wurde ein NcoI-BsaBI-Fragment des Plasmides pACYC184-LH-cysEX-orf306 (beschrieben in
Der Ligationsansatz wurde in "DH5α™-T1R E. coli-Zellen" (Life Technologies GmbH) transformiert, in diesen Zellen vermehrt und die DNA-Sequenz der isolierten Plasmide mittels Sequenzierung verifiziert. Das resultierende Expressionsplasmid trägt die Bezeichnung pcysEX-GAPDH-ORF306_tetR (siehe
Als Ausgangsplasmide für die Klonierung und Expression des Cyclodextrin-Glycosyl-Transferase (CGTase)-Gens aus Klebsiella pneumoniae M5al (Genbanknr. M15264) dienten erneut das Plasmid pMT1 sowie das in
Der Ligationsansatz wurde in "DH5α™-T1R E. coli-Zellen" (Life Technologies GmbH) transformiert, in diesen Zellen vermehrt und die DNA-Sequenz der isolierten Plasmide mittels Sequenzierung verifiziert. Das resultierende Expressionsplasmid trägt die Bezeichnung pCGT_tetR (siehe
Als Ausgangsplasmide für die Klonierung und Expression der Gene des Anti-Lysozym-Fab-Fragments dienten erneut das Plasmid pMT1 sowie das in
Der Ligationsansatz wurde in "DH5α™-T1R E. coli-Zellen" (Life Technologies GmbH) transformiert, in diesen Zellen vermehrt und Fragment des Plasmides pMT1 kodiert für den ColE1-ori, den lac/tac-Operator und das Tetracylinresistenzgen (tetR).The ligation mixture was transformed into "DH5α ™ -T1R E. coli cells" (Life Technologies GmbH), and multiplied in these cells Fragment of the plasmid pMT1 codes for the ColE1-ori, the lac / tac operator and the tetracycline resistance gene ( tetR ).
Der Ligationsansatz wurde in "DH5α™-T1R E. coli-Zellen" (Life Technologies GmbH) transformiert, in diesen Zellen vermehrt und die DNA-Sequenz der isolierten Plasmide mittels Sequenzierung verifiziert. Das resultierende Expressionsplasmid trägt die Bezeichnung pFab-anti-Lysozym_tetR (siehe
Als Ausgangsplasmide für die Generierung von Produktionsplasmiden mit pyrH oder plsC als Markergen dienten die in den Beispielen 5 bis 7 beschriebenen Plasmide pcysEX-GAPDH-ORF306_tetR, pCGT_tetR und pFab-anti-Lysozym_tetR. All diese Plasmide besitzen eine einzelne NcoI-Restriktionsschnittstelle (siehe Abbildungen 5 bis 7). Diese universelle NcoI-Schnittstelle wurde für die Klonierung bzw. Integration der Gene pyrH oder plsC genutzt.The plasmids pcysEX-GAPDH-ORF306_tetR, pCGT_tetR and pFab-anti-Lysozyme_tetR, described in Examples 5 to 7, served as starting plasmids for the generation of production plasmids with pyrH or plsC as marker gene. All of these plasmids have a single NcoI restriction site (see Figures 5 to 7). This universal NcoI interface was used for the cloning or integration of the genes pyrH or plsC .
Hierfür wurden die in Beispiel 1 beschriebenen, und mit dem Restriktionsenzym NcoI geschnittenen PCR-Produkte mit den Plasmiden pcysEX-GAPDH-ORF306_tetR, pCGT_tetR und pFab-anti-Lysozyme_tetR ligiert, nachdem diese ebenfalls mit NcoI geschnitten wurden. Die einzelnen Ligationsansätze wurden in "DH5α™-T1R E. coli-Zellen" (Life Technologies GmbH) transformiert, in diesen Zellen vermehrt und die DNA-Sequenz der isolierten Plasmide mittels Sequenzierung verifiziert. Die resultierenden Konstrukte tragen, je nach Orientierung und verwendeten Markergen, folgende Bezeichnungen:
- pcysEX-GAPDH-ORF306_pyrH1_tetR (siehe
Fig. 8 ) und pcysEX-GAPDH-ORF306_pyrH2_tetR - pCGT_pyrH1_tetR (siehe
Fig. 9 )und pCGT_pyrH2_tetR - pFab-anti-Lysozyme_pyrH1_tetR (siehe
Fig. 10 ) und pFab-anti-Lysozyme_pyrH2_tetR
- pcysEX-GAPDH-ORF306_pyrH1_tetR (see
Fig. 8 ) and pcysEX-GAPDH-ORF306_pyrH2_tetR - pCGT_pyrH1_tetR (see
Fig. 9 ) and pCGT_pyrH2_tetR - pFab-anti-Lysozyme_pyrH1_tetR (see
Fig. 10 ) and pFab-anti-Lysozyme_pyrH2_tetR
Für die finale Entfernung des tetR-Gens wurde mit den Varianten pcysEX-GAPDH-ORF306_pyrH1_tetR, pCGT_pyrH1_tetR, pFab-anti-Lysozyme_pyrH1_tetR, pcysEX-GAPDH-ORF306_plsC1_tetR, pCGT_plsC1_tetR und pFab-anti-Lysozyme_plsC1_tetR weitergearbeitet (siehe
Als Ausgangsplasmide für die Generierung von Produktionsplasmiden ohne das Antibiotikaresistenzgen tetR und mit pyrH oder plsC als Markergen dienten die in Beispiel 8 beschriebenen Plasmide pcysEX-GAPDH-ORF306_pyrH1_tetR, pCGT_pyrH1_tetR, pFab-anti-Lysozyme_pyrH1_tetR, pcysEX-GAPDH-ORF306_ plsC1_tetR, pCGT_plsC1_tetR und pFab-anti-Lysozyme_ plsC1_tetR. Die Entfernung des Antibiotikaresistenzgens tetR von den in Beispiel 8 beschriebenen Plasmiden pFab-anti-Lysozyme_pyrH1_tetR und pFab-anti-Lysozym_plsC1_tetR erfolgte über einen Verdau mit dem Restriktionsenzym ClaI und anschließender Religation.As starting plasmids for the generation of production plasmids without the antibiotic resistance gene tetR and with pyrH or plsC as marker gene, the plasmids pcysEX-GAPDH-ORF306_pyrH1_tetR, pCGT_pyrH1_tetR, pFab-anti-tet_tetR1, pFab-anti-tet_tetC1, pFab-anti-tet_tetC1, pFab-anti-tet_tetC1, pFab-anti-tet_tetC1, pFab-anti-tet_tetC6, pDRH1_FetCF6, pDRH1_tetCF6, pDRH1_tetRs anti-Lysozyme_ plsC1_tetR. The antibiotic resistance gene tetR was removed from the plasmids pFab-anti-Lysozyme_pyrH1_tetR and pFab-anti-Lysozyme_plsC1_tetR described in Example 8 via digestion with the restriction enzyme ClaI and subsequent religation.
Für die Plasmide pcysEX-GAPDH-ORF306_pyrH1_tetR bzw. pcysEX-GAPDH-ORF306_plsC1_tetR wurde ähnlich verfahren, nur dass das Gen tetR über einen Partialverdau mit ClaI entfernt wurde, da sich zwei weitere ClaI-Schnittstellen in den Strukturgenen cysEX und orf306 befinden (siehe
Im Falle von pCGT_plsC1_tetR wurde das tetR-Gen über einen Verdau mit den Enzymen StuI (schneidet blunt End) und FspI (schneidet blunt End) vom Plasmid entfernt.In the case of pCGT_plsC1_tetR, the tetR gene was removed from the plasmid by digestion with the enzymes StuI (cuts blunt end) and FspI (cuts blunt end).
Im Falle von pCGT_pyrH1_tetR wurde das tetR-Gen ebenfalls über einen Partialverdau mit den Enzymen StuI (schneidet blunt End) und FspI (schneidet blunt End) entfernt, da sich eine weitere FspI-Schnittstelle im pyrH-Gen befindet (siehe
Die jeweiligen linearen Vektorfragmente ohne tetR wurden nach dem Restriktionsverdau über eine Agarose-Gelelektrophorese gereinigt und mit dem "QIAquick Gel Extraction Kit" (Qiagen und FspI (schneidet blunt End) entfernt, da sich eine weitere FspI-Schnittstelle im pyrH-Gen befindet (siehe
Die jeweiligen linearen Vektorfragmente ohne tetR wurden nach dem Restriktionsverdau über eine Agarose-Gelelektrophorese gereinigt und mit dem "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, D) nach Angaben des Herstellers aus dem Agarosegel isoliert. Anschließend wurde das jeweilige tetRfreie Vektorfragment religiert.The respective linear vector fragments without tetR were purified by agarose gel electrophoresis after restriction digestion and isolated from the agarose gel using the "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, D) according to the manufacturer's instructions. The respective tetR- free vector fragment was then religated.
Die entsprechenden Ligationsansätze wurden mit einer abgewandelten CaCl2-Methode in die im Beispiel 4 beschriebenen Stämme W3110ΔpyrH/pAF-ts-pyrH bzw. W31101pp3ΔpyrH/pAF-ts-pyrH oder W3110ΔpyrH/pAF-ts-plsC bzw. W31101pp3ΔpyrH/pAF-ts-plsC transformiert. Für die Transformation, d.h. die Einbringung der Antibiotikaresistenz-freien Produktionsplasmide mit pyrH oder plsC als Markergen in E. coli Stämme mit entsprechender chromosomaler Deletion (pyrH oder plsC) wurde folgendermaßen verfahren:
Nach der Zugabe von 5 bis 20 µl des jeweiligen Ligationsansatzes zu 100 µl CaCl2-kompetenter Zellen der Stämme W3110ΔpyrH/pAF-ts-pyrH und W3110lpp3ΔpyrH/pAF-ts-pyrH bzw. W3110ΔplsC/pAF-ts-plsC und W3110lpp3ΔplsC/pAF-ts-plsC wurden die Zellen für weitere 30 Minuten auf Eis inkubiert. Nach einem kurzzeitigen Hitzeschock für 45 Sekunden bei 42°C wurden die Zellen 2 min auf Eis abgekühlt. Anschließend wurden dem Transformationsansatz 900 µl LB-Medium zugesetzt und die Zellen, nicht wie üblich bei 37°C, sondern 30 bis 90 min bei 47°C bis 55°C inkubiert / regeneriert. Die weitere Inkubation bei erhöhter nicht permissiver Temperatur erfolgte dann für 15 bis 24 h bei 40-45°C auf LB-Agarplatten oder in flüssigem LB-Medium ohne Antibiotikum (z.B. ohne Tetracyclin).The corresponding ligation batches were converted into the strains W3110ΔpyrH / pAF-ts-pyrH and W31101pp3ΔpyrH / pAF-ts-pyrH or W3110ΔpyrH / pAF-ts-plsC or W31101pp3Δpyrs- / pAF-tsC described in Example 4 using a modified CaCl 2 method plsC transformed. For the transformation, ie the introduction of the antibiotic resistance-free production plasmids with pyrH or plsC as marker gene in E. coli strains with a corresponding chromosomal deletion ( pyrH or plsC ), the following procedure was used:
After adding 5 to 20 µl of the respective ligation batch to 100 µl CaCl 2 -competent cells of the strains W3110ΔpyrH / pAF-ts-pyrH and W3110lpp3ΔpyrH / pAF-ts-pyrH or W3110ΔplsC / pAF-ts-plsC and W3110lpp3ΔplsC and With ts-plsC, the cells were incubated on ice for a further 30 minutes. After a brief heat shock for 45 seconds at 42 ° C., the cells were cooled on ice for 2 minutes. Then 900 μl of LB medium were added to the transformation mixture and the cells were incubated / regenerated not as usual at 37 ° C., but for 30 to 90 minutes at 47 ° C. to 55 ° C. The further incubation at an elevated non-permissive temperature then took place for 15 to 24 hours at 40-45 ° C. on LB agar plates or in liquid LB medium without antibiotic (for example without tetracycline).
Die 30 - 90 minütige Regenerationsphase bei 47°C bis 55°C sowie die Kultivierung für 15 bis 24 h bei erhöhter, nicht permissiver Temperatur erleichtert den Austausch der temperatursensitiven Plasmide pAF-ts-pyrH bzw. pAF-ts-plsC gegen das finale, Antibiotikaresistenz-freie Produktionsplasmid mit pyrH oder plsC als neuem Selektionsmarkergen.The 30-90 minute regeneration phase at 47 ° C to 55 ° C and cultivation for 15 to 24 h at an elevated, non-permissive temperature facilitate the exchange of the temperature-sensitive plasmids pAF-ts-pyrH or pAF-ts-plsC for the final, Antibiotic resistance-free production plasmid with pyrH or plsC as a new selection marker gene.
Die besten Transformations-Ergebnisse wurden erzielt, wenn die transformierten Zellen 60 min bei 52°C regeneriert und anschließend 20 h bei 42°C auf LB-Agarplatten inkubiert wurden. Eine Vorab-Selektion auf Verlust des temperatursensitiven Plasmids pAF-ts-pyrH bzw. pAF-ts-plsC bei gleichzeitigem Austausch gegen das jeweilige pyrH- oder plsC-haltige Produktionsplasmid ohne Tetracyclinresistenzgen tetR erfolgte zunächst auf LB-Agarplatten ohne Antibiotikum.The best transformation results were achieved when the transformed cells were regenerated at 52 ° C. for 60 minutes and then incubated on LB agar plates at 42 ° C. for 20 hours. A preliminary selection for loss of the temperature-sensitive plasmid pAF-ts-pyrH or pAF-ts-plsC with simultaneous exchange for the respective pyrH- or plsC-containing production plasmid without the tetracycline resistance gene tetR was initially carried out on LB agar plates without antibiotic.
Anschließend wurden die vorselektierten Bakterien-Klone auf Ampicillin-Sensitivität, d.h. den Verlust des temperatursensitiven Plasmides pAF-ts-pyrH bzw. pAF-ts-plsC überprüft.The preselected bacterial clones were then checked for ampicillin sensitivity, i.e. the loss of the temperature-sensitive plasmid pAF-ts-pyrH or pAF-ts-plsC.
Die pyrH- oder plsC-kodierenden Produktionsplasmide aus Ampicillin-sensitiven Klonen wurden abschließend mittels Restriktionsverdau überprüft. Restriktionskarten der pyrH-kodierenden Produktionsplasmide pcysEX-GAPDH-ORF306_pyrH, pCGT_pyrH und pFab-anti-Lysozyme_pyrH, jeweils ohne das Antibiotikaresistenzgen tetR, sind in den Abbildungen 14 bis 16 dargestellt. Restriktionskarten der plsC-kodierenden Produktionsplasmide pcysEX-GAPDH-ORF306_plsC, pCGT_plsC und pFab-anti-Lysozyme_plsC, jeweils ohne das Antibiotikaresistenzgen tetR, sind in den Abbildungen 17 bis 19 dargestellt.The production plasmids encoding pyrH or plsC from ampicillin-sensitive clones were then checked by means of restriction digestion. Restriction maps of the pyrH -coding production plasmids pcysEX-GAPDH-ORF306_pyrH, pCGT_pyrH and pFab-anti-Lysozyme_pyrH, each without the antibiotic resistance gene tetR , are shown in FIGS. 14 to 16. Restriction maps of the plsC -coding production plasmids pcysEX-GAPDH-ORF306_plsC, pCGT_plsC and pFab-anti-Lysozyme_plsC, each without the antibiotic resistance gene tetR, are shown in Figures 17 to 19.
Die auf diese Art und Weise generierten und überprüften Antibiotikaresistenz-freien E. coli-Stämme mit pyrH- oder plsCkodierendem Produktionsplasmid und chromosomaler pyrH- bzw. plsC-Deletion tragen die Bezeichnungen:
- W3110ΔpyrH/pcysEX-GAPDH-ORF306_pyrH
- W3110lpp3ΔpyrH/pCGT_pyrH
- W3110lpp3ΔpyrH/pFab-anti-Lysozym_pyrH
- W3110ΔplsC/pcysEX-GAPDH-ORF306_plSC
- W3110lpp3ΔplsC/pCGT_plsC
- W3110lpp3ΔplsC/pFab-anti-Lysozym_plsC
- W3110ΔpyrH / pcysEX-GAPDH-ORF306_pyrH
- W3110lpp3ΔpyrH / pCGT_pyrH
- W3110lpp3ΔpyrH / pFab-anti-lysozyme_pyrH
- W3110ΔplsC / pcysEX-GAPDH-ORF306_plSC
- W3110lpp3ΔplsC / pCGT_plsC
- W3110lpp3ΔplsC / pFab-anti-lysozyme_plsC
Anschließend wurde die Vorkultur 1 vollständig in 100 ml SM1-Medium überführt (12 g/l K2HPO4, 3 g/l KH2PO4, 5 g/l (NH4)2SO4, 0,3 g/l MgSO4 x 7 H2O, 0,015 g/l CaCl2 x 2 H2O, 0,002 g/l FeSO4 x 7 H2O, 1 g/l Na2Citrat x 2 H2O, 0,1 g/l NaCl, 1 ml/l Spurenelementlösung, bestehend aus 0,15 g/l Na2MoO4 x 2H2O, 2,5 g/l H3BO3, 0,7 g/l CoCl2 x 6 H2O, 0,25 g/l CuSO4 x 5 H2O, 1,6 g/l MnCl2 x 4 H2O, 0,3 g/l ZnSO4 x 7 H2O), das mit 5 g/l Glucose und 5 mg/l Vitamin B1 supplementiert war. Die Kulturen wurden in Erlenmeyerkolben (1 1) bei 30 °C für 17 h mit 150 rpm geschüttelt. Nach dieser Inkubation lag die optische Dichte bei 600 nm (OD600) zwischen 3 und 5. Für die Kultivierung von W3110/pcysEX-GAPDH-ORF306_tetR im Sinne des Stands der Technik, d.h. mit Antibiotikum als Selektionsmittel, wurde das Medium mit 15 mg/L Tetracyclin supplementiert.Subsequently, the preculture 1 was completely transferred into 100 ml of SM1 medium (12 g / l K 2 HPO4, 3 g / l KH 2 PO 4 , 5 g / l (NH 4 ) 2SO 4 , 0.3 g / l MgSO 4 x 7 H 2 O, 0.015 g / l CaCl 2 x 2 H 2 O, 0.002 g / l FeSO 4 x 7 H 2 O, 1 g / l Na 2 citrate x 2 H 2 O, 0.1 g / l NaCl , 1 ml / l trace element solution, consisting of 0.15 g / l Na 2 MoO 4 x 2H 2 O, 2.5 g / l H 3 BO 3 , 0.7 g / l CoCl 2 x 6 H 2 O, 0 , 25 g / l CuSO 4 x 5 H 2 O, 1.6 g / l MnCl 2 x 4 H 2 O, 0.3 g / l ZnSO 4 x 7 H 2 O), which contains 5 g / l glucose and 5 mg / l vitamin B1 was supplemented. The cultures were shaken in Erlenmeyer flasks (11) at 30 ° C. for 17 hours at 150 rpm. After this incubation, the optical density at 600 nm (OD 600 ) was between 3 and 5. For the cultivation of W3110 / pcysEX-GAPDH-ORF306_tetR in the sense of the prior art, ie with an antibiotic as a selection agent, the medium was given 15 mg / L supplemented with tetracycline.
Die Fermentation wurde in Fermentern des Typs BIOSTAT B der Firma Sartorius Stedim durchgeführt. Es wurde ein Kulturgefäß mit 2 1 Gesamtvolumen verwendet. Das Fermentationsmedium (900 ml) enthält 15 g/l Glucose, 10 g/l Trypton (Difco), 5 g/l Hefeextrakt (Difco), 5 g/l (NH4)2SO4, 1,5 g/l KH2PO4, 0,5 g/l NaCl, 0,3 g/l MgSO4 x 7 H2O, 0,015 g/l CaCl2 x 2 H2O, 0,075 g/l FeSO4 x 7 H2O, 1 g/l Na2Citrat x 2 H2O und 1 ml Spurenelementlösung (siehe oben) und 0,005 g/l Vitamin B1. Der pH-Wert im Fermenter wurde zu Beginn durch Zupumpen einer 25% NH4OH-Lösung auf 6,5 eingestellt. Während der Fermentation wurde der pH-Wert durch automatische Korrektur mit 25% NH4OH auf einem Wert von 6,5 gehalten. Zum Animpfen wurden 100 ml der Vorkultur 2 in das Fermentergefäß gepumpt. Das Anfangsvolumen betrug somit etwa 1 1. Die Kulturen wurden zu Beginn mit 400 rpm gerührt und mit 2 vvm einer über einen Sterilfilter entkeimten Druckluft begast. Unter diesen Startbedingungen war die Sauerstoff-Sonde vor der Inokulation auf 100% Sättigung kalibriert worden. Der Soll-Wert für die O2-Sättigung während der Fermentation wurde auf 50 % eingestellt. Nach Absinken der O2-Sättigung unter den Soll-Wert wurde eine Regulationskaskade gestartet, um die O2-Sättigung wieder an den Soll-Wert heranzuführen. Dabei wurde zunächst die Gaszufuhr kontinuierlich erhöht (auf max. 5 vvm) und anschließend die Rührgeschwindigkeit (auf max. 1.500 rpm) kontinuierlich gesteigert.The fermentation was carried out in fermenters of the type BIOSTAT B from Sartorius Stedim. A culture vessel with a total volume of 2 liters was used. The fermentation medium (900 ml) contains 15 g / l glucose, 10 g / l tryptone (Difco), 5 g / l yeast extract (Difco), 5 g / l (NH 4 ) 2 SO 4 , 1.5 g / l KH 2 PO 4 , 0.5 g / l NaCl, 0.3 g / l MgSO 4 x 7 H 2 O, 0.015 g / l CaCl 2 x 2 H 2 O, 0.075 g / l FeSO 4 x 7 H 2 O, 1 g / l Na 2 citrate x 2 H 2 O and 1 ml trace element solution (see above) and 0.005 g / l vitamin B1. The pH in the fermenter was initially adjusted to 6.5 by pumping in a 25% NH4OH solution. During the fermentation, the pH was kept at a value of 6.5 by automatic correction with 25% NH 4 OH. For inoculation, 100 ml of preculture 2 were pumped into the fermenter vessel. The initial volume was thus about 11. The cultures were initially stirred at 400 rpm and gassed with 2 vvm of compressed air disinfected via a sterile filter. Under these starting conditions, the oxygen probe was calibrated to 100% saturation before the inoculation. The target value for the O 2 saturation during the fermentation was set to 50%. After the O 2 saturation had fallen below the target value, a regulation cascade was started in order to bring the O 2 saturation back to the target value. First the Gas supply increased continuously (to max. 5 vvm) and then the stirring speed (to max. 1,500 rpm) increased continuously.
Die Fermentation wurde bei einer Temperatur von 30 °C durchgeführt. Nach 2 h Fermentationszeit erfolgte die Zufütterung einer Schwefelquelle in Form einer sterilen 60 % Natrium-Thiosulfat x 5 H2O - Stammlösung mit einer Rate von 1,5 ml pro Stunde. Sobald der Glukose-Gehalt im Fermenter von anfänglich 15 g/l auf ca. 2 g/l abgesunken war, erfolgte eine kontinuierliche Zudosierung einer 56% Glukose-Lösung. Die Fütterungsrate wurde so eingestellt, dass die Glukosekonzentration im Fermenter 2 g/l fortan nicht mehr überstieg.The fermentation was carried out at a temperature of 30 ° C. After a fermentation time of 2 h, a sulfur source was fed in in the form of a sterile 60% sodium thiosulfate x 5 H 2 O stock solution at a rate of 1.5 ml per hour. As soon as the glucose content in the fermenter had fallen from initially 15 g / l to approx. 2 g / l, a 56% glucose solution was continuously metered in. The feeding rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g / l.
Die Glukose-Bestimmung wurde mit einem Glukoseanalysator der Firma YSI (Yellow Springs, Ohio, USA) durchgeführt. Für die Fermentation von Konstrukt W3110/pcysEX-GAPDH-ORF306_tetR im Sinne des Stands der Technik, d.h. mit Antibiotikum als Selektionsmittel, wurde das Medium mit 15 mg/L Tetracyclin supplementiert.The glucose determination was carried out with a glucose analyzer from YSI (Yellow Springs, Ohio, USA). For the fermentation of construct W3110 / pcysEX-GAPDH-ORF306_tetR in the sense of the prior art, i.e. with an antibiotic as selection agent, the medium was supplemented with 15 mg / L tetracycline.
Die Fermentationsdauer betrug 48 Stunden. Danach wurden Proben entnommen und der Gehalt von L-Cystein und den davon abgeleiteten Derivaten im Kulturüberstand (v.a. L-Cystein und Thiazolidin) und im Niederschlag (L-Cystin) jeweils getrennt voneinander bestimmt. Zu diesem Zweck wurde jeweils der colorimetrische Test von Gaitonde verwendet (
L-Cystein im Kulturüberstand und L-Cystin im Niederschlag. Dabei entspricht jedes Molekül L-Cystin zwei Molekülen L-Cystein.
**erfindungsgemäßes Konstrukt
** inventive construct
Mit Hilfe einer 1pp-Mutante von E. coli können biotechnologisch relevante Enzyme wie beispielsweise CGTasen produziert und ins Medium sekretiert werden (
Die sekretorische Produktion der CGTase erfolgte in 10 L Rührkesselfermentern mit den Stämmen W3110lpp3/pCGT_tetR (Kontrolle), W3110lpp3ΔpyrH/pCGT_pyrH und W3110lpp3ΔpyrH/pCGT_plsC. Der mit 6 l des Fermentationsmediums FM4 (1,5 g/l KH2PO4; 5 g/l (NH4) 2SO4; 0, 5 g/l MgSO4 x 7 H2O; 0,15 g/l CaCl2 x 2 H2O, 0,075 g/l FeSO4 x 7 H2O; 1 g/l Na2Citrat x 2 H2O; 0,5 g/l NaCl; 1 ml/1 Spurenelementlösung (0,15 g/l Na2MoO4 x 2 H2O; 2,5 g/l Na3BO3; 0,7 g/l CoCl2 x 6 H2O; 0,25 g/l CuSO4 x 5 H2O; 1,6 g/l MnCl2 x 4 H2O; 0,3 g/l ZnSO4 x 7 H2O); 5 mg/l Vitamin B1; 3 g/l Phyton; 1,5 g/l Hefeextrakt; 10 g/l Glukose) befüllte Fermenter wurde im Verhältnis 1:10 mit einer Vorkultur, die über Nacht im glei-Für die Fermentation des Konstruktes W31101pp3/pCGT_tetR im Sinne des Stands der Technik, d.h. mit Antibiotikum als Selektionsmittel, wurde das Medium mit 15 mg/L Tetracyclin supplementiert.The secretory production of CGTase took place in 10 L stirred tank fermenters with the strains W3110lpp3 / pCGT_tetR (control), W3110lpp3ΔpyrH / pCGT_pyrH and W3110lpp3ΔpyrH / pCGT_plsC. The one with 6 l of fermentation medium FM4 (1.5 g / l KH 2 PO 4 ; 5 g / l (NH 4 ) 2 SO 4 ; 0.5 g / l MgSO 4 x 7 H 2 O; 0.15 g / l CaCl 2 x 2 H 2 O, 0.075 g / l FeSO 4 x 7 H 2 O; 1 g / l Na 2 citrate x 2 H 2 O; 0.5 g / l NaCl; 1 ml / 1 trace element solution (0, 15 g / l Na 2 MoO 4 x 2 H 2 O; 2.5 g / l Na 3 BO 3 ; 0.7 g / l CoCl 2 x 6 H 2 O; 0.25 g / l CuSO 4 x 5 H 2 O; 1.6 g / l MnCl 2 x 4 H 2 O; 0.3 g / l ZnSO 4 x 7 H 2 O); 5 mg / l vitamin B 1 ; 3 g / l phyton; 1.5 g / l yeast extract; 10 g / l glucose) filled fermenter in a ratio of 1:10 with a preculture, which overnight in the same for the fermentation of the construct W31101pp3 / pCGT_tetR in the sense of the prior art, ie with antibiotic as selection agent, the medium was supplemented with 15 mg / L tetracycline.
Nach 72 h Fermentation wurden Proben entnommen, die Zellen durch Zentrifugation vom Fermentationsmedium abgetrennt und der CGTase-Gehalt im Fermentationsüberstand, wie in Beispiel 4 von
In Tabelle 2 sind die Ausbeuten an funktioneller CGTase sowie die Aktivitäten im Fermentationsüberstand aufgelistet.
**erfindungsgemäßes Konstrukt
** inventive construct
Mit Hilfe einer lpp-Mutante von E. coli können auch funktionelle Fab-Antikörperfragmente extrazellulär hergestellt werden (
Das vorliegende Beispiel beschreibt die Produktion eines Fab-Fragments des gut charakterisierten Anti-Lysozym-Antikörpers D1.3.
Die Plasmide pFab-anti-Lysozym_tetR, pFab-anti-Lysozym_pyrH und pFab-anti-Lysozym_plsC enthalten neben den Markergenen tetR, pyrH bzw. plsC u.a. auch die Strukturgene für die HC und die LC des Fab-Fragments in Form eines Operons. Dabei ist die HC in frame an das 3'-Ende der ompA-Signalsequenz (ompAss) und die LC in frame an das 3'-Ende einer CGTase-Signalsequenz (cgtss) anfusioniert. Die Expression des ompAss-HC-cgtss-LC-Operons steht unter der Kontrolle des tac-Promotors.The present example describes the production of a Fab fragment of the well characterized anti-lysozyme antibody D1.3.
The plasmids pFab-anti-Lysozym_tetR, pFab-anti-Lysozym_pyrH and pFab-anti-Lysozym_plsC contain not only the marker genes tetR, pyrH or plsC , but also the structural genes for the HC and LC of the Fab fragment in the form of an operon. The HC in frame is fused to the 3 'end of the ompA signal sequence (ompA ss ) and the LC in frame to the 3' end of a CGTase signal sequence (cgt ss ). The expression of the ompA ss -HC-cgt ss -LC operon is under the control of the tac promoter.
Die Produktion des Anti-Lysozym-Fab-Fragments im 10 l-Maßstab erfolgte analog dem in Beispiel 11 anhand der CGTase beschriebenen Verfahren mit den Stämmen W3110lpp3/pFab-anti-Lysozym_tetR, W3110lpp3ΔpyrH/pFab-anti-Lysozym_pyrH und W3110lpp3ΔplsC/pFab-anti-Lysozym_plsC. Für die Fermentation von E. coli W3110lpp3/pFab-anti-Lysozym_tetR im Sinne des Stands der Technik, d.h. mit Antibiotikum als Selektionsmittel, wurde das Medium mit 15 mg/L Tetracyclin supplementiert.The anti-lysozyme Fab fragment was produced on a 10 l scale analogously to the method described in Example 11 using CGTase with the strains W3110lpp3 / pFab-anti-lysozyme_tetR, W3110lpp3ΔpyrH / pFab-anti-Lysozyme_pyrH and W3110lpp3ΔplsC / pFab-anti-pFab -Lysozyme_plsC. For the fermentation of E. coli W3110lpp3 / pFab-anti-Lysozyme_tetR in the sense of the prior art, ie with antibiotic as selection agent, the medium was supplemented with 15 mg / L tetracycline.
Nach 72 h Fermentation wurden Proben entnommen und anschließend die Zellen durch Zentrifugation vom Fermentationsmedium abgetrennt.After 72 hours of fermentation, samples were taken and then the cells were separated from the fermentation medium by centrifugation.
Die Reinigung des Anti-Lysozym-Fab-Fragments aus den Fermentationsüberstanden erfolgte mittels Affinitätschromatographie wie in
Die Quantifizierung sowie die Bestimmung der Aktivität des gereinigten Anti-Lysozym-Fab-Fragments erfolgte über einen ELISA-Test mit Lysozym als Antigen (
In Tabelle 2 sind die hochgerechneten Ausbeuten an funktionellem Anti-Lysozym-Fab-Fragment im Fermentationsüberstand aufgelistet, basierend auf isolierten Mengen aus jeweils 20 ml Fermentationsüberstand nach 72 h Fermentation.Table 2 lists the extrapolated yields of functional anti-lysozyme Fab fragment in the fermentation supernatant, based on isolated amounts from each 20 ml fermentation supernatant after 72 h fermentation.
Die Quantifizierung sowie die Bestimmung der Aktivität des gereinigten Anti-Lysozym-Fab-Fragments erfolgte über einen ELISA-Test mit Lysozym als Antigen (
In Tabelle 2 sind die hochgerechneten Ausbeuten an funktionellem Anti-Lysozym-Fab-Fragment im Fermentationsüberstand aufgelistet, basierend auf isolierten Mengen aus jeweils 20 ml Fermentationsüberstand nach 72 h Fermentation.
**erfindungsgemäßes Konstrukt
** inventive construct
Die Plasmidstabilität wurde mittels Plasmidpräparation mit anschließendem Restriktionsverdau überprüft. Hierfür wurden nach Beendigung der Kultivierung der Produktionsstämme (z.B. nach 72 h Fermentation) verschiedene Verdünnungen der Kulturen auf LB-Agarplatten ausplattiert. Für die anschließende Ermittlung der Plasmidstabilität, d.h. die Identifizierung Plasmid-tragender Zellen (Kolonien) wurden nur LB-Platten mit Einzelkolonien für die Auswertung herangezogen.The plasmid stability was checked by means of plasmid preparation with subsequent restriction digestion. For this purpose, after the cultivation of the production strains had ended (eg after 72 h fermentation), various dilutions of the cultures were plated out on LB agar plates. For the subsequent determination of the plasmid stability, ie the identification of plasmid-carrying Cells (colonies) were only used LB plates with single colonies for the evaluation.
Insgesamt wurden 50 einzelne Kolonien in flüssigem LB-Medium für 15 bis 20 Stunden kultiviert und anschließend Plasmid-DNA aus diesen Kulturen isoliert. Anhand charakteristischer Restriktionsmuster für die einzelnen Produktionsplasmide wurde die Richtigkeit der isolierten Plasmide verifiziert.A total of 50 individual colonies were cultivated in liquid LB medium for 15 to 20 hours and then plasmid DNA was isolated from these cultures. The correctness of the isolated plasmids was verified on the basis of characteristic restriction patterns for the individual production plasmids.
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<110> Consortium für elektrochemische Industrie - Wacker Chemie AG
Bornhövd, Dr. Carsten
Thön, Dr. Marcel<110> Consortium for the electrochemical industry - Wacker Chemie AG
Bornhövd, Dr. Carsten
Thön, Dr. Marcel - <120> Mikroorganismenstamm und Verfahren zur antibiotikafreien, fermentativen Herstellung von niedermolekularen Substanzen und Proteinen<120> Microorganism strain and process for antibiotic-free, fermentative production of low-molecular substances and proteins
- <130> Co11517<130> Co11517
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<150> Co11517
<151> 2015-08-27<150> Co11517
<151> 2015-08-27 - <160> 17<160> 17
- <170> PatentIn version 3.5<170> Patent In version 3.5
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<210> 1
<211> 726
<212> DNA
<213> Escherichia coli<210> 1
<211> 726
<212> DNA
<213> Escherichia coli -
<220>
<221> gene
<222> (1)..(726)
<223> pyrH<220>
<221> genes
<222> (1) .. (726)
<223> pyrH - <400> 1 <400> 1
-
<210> 2
<211> 241
<212> PRT
<213> Escherichia coli<210> 2
<211> 241
<212> PRT
<213> Escherichia coli -
<220>
<221> PEPTIDE
<222> (1)..(241)<220>
<221> PEPTIDES
<222> (1) .. (241) - <400> 2 <400> 2
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<210> 3
<211> 738
<212> DNA
<213> Escherichia coli<210> 3
<211> 738
<212> DNA
<213> Escherichia coli -
<220>
<221> gene
<222> (1)..(738)
<223> plsC<220>
<221> genes
<222> (1) .. (738)
<223> plsC - <400> 3 <400> 3
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<210> 4
<211> 245
<212> PRT
<213> Escherichia coli<210> 4
<211> 245
<212> PRT
<213> Escherichia coli -
<220>
<221> PEPTIDE
<222> (1)..(245)<220>
<221> PEPTIDES
<222> (1) .. (245) -
<220>
<221> PEPTIDE
<222> (1)..(245)
<223> PlsC<220>
<221> PEPTIDES
<222> (1) .. (245)
<223> PlsC - <400> 4 <400> 4
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<210> 5
<211> 40
<212> DNA
<213> synthetisches Oligonukleotid<210> 5
<211> 40
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: pyrH-NcoI-fw<220>
<221> misc_feature
<223> oligonucleotide: pyrH-NcoI-fw -
<400> 5
ccccccatgg ccatcttgta aattcagcta acccttgtgg 40<400> 5
ccccccatgg ccatcttgta aattcagcta acccttgtgg 40 -
<210> 6
<211> 40
<212> DNA
<213> synthetisches Oligonukleotid<210> 6
<211> 40
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc _feature
<223> Oligonukleotid: pyrH-NcoI-rev<220>
<221> misc _feature
<223> Oligonucleotide: pyrH-NcoI-rev -
<400> 6
ggggccatgg atcctcacgt acttttgtac gctccggttg 40<400> 6
ggggccatgg atcctcacgt acttttgtac gctccggttg 40 -
<210> 7
<211> 37
<212> DNA
<213> synthetisches Oligonukleotid<210> 7
<211> 37
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: plsC-NcoI-fw<220>
<221> misc_feature
<223> oligonucleotide: plsC-NcoI-fw -
<400> 7
ccccccatgg atttgctcca tgtaaaactg gctaaag 37<400> 7
ccccccatgg atttgctcca tgtaaaactg gctaaag 37 -
<210> 8
<211> 40
<212> DNA
<213> synthetisches Oligonukleotid<210> 8
<211> 40
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: plsC-NcoI-rev<220>
<221> misc_feature
<223> oligonucleotide: plsC-NcoI-rev -
<400> 8
ggggccatgg tgaaaccgtt gtttattcat gcgttgcgat 40<400> 8
ggggccatgg tgaaaccgtt gtttattcat gcgttgcgat 40 -
<210> 9
<211> 71
<212> DNA
<213> synthetisches Oligonukleotid<210> 9
<211> 71
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> synthetisches Oligonukleotid: pyrH-fw<220>
<221> misc_feature
<223> synthetic oligonucleotide: pyrH-fw -
<220>
<221> misc_feature
<223> Oligonukleotid: pyrH-fw<220>
<221> misc_feature
<223> oligonucleotide: pyrH-fw - <400> 9 <400> 9
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<210> 10
<211> 70
<212> DNA
<213> synthetisches Oligonukleotid<210> 10
<211> 70
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: pyrH-rev<220>
<221> misc_feature
<223> oligonucleotide: pyrH-rev - <400> 10 <400> 10
-
<210> 11
<211> 30
<212> DNA
<213> synthetisches Oligonukleotid<210> 11
<211> 30
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: pyrH-check-for<220>
<221> misc_feature
<223> oligonucleotide: pyrH-check-for -
<400> 11
gcataacgct gaagtgactg gcttcatccg 30<400> 11
gcataacgct gaagtgactg gcttcatccg 30 -
<210> 12
<211> 30
<212> DNA
<213> synthetisches Oligonukleotid<210> 12
<211> 30
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: pyrH-check-rev<220>
<221> misc_feature
<223> oligonucleotide: pyrH-check-rev -
<400> 12
cagaccagac agtcacacac agtggaagtg 30<400> 12
cagaccagac agtcacacac agtggaagtg 30 -
<210> 13
<211> 71
<212> DNA
<213> synthetisches Oligonukleotid<210> 13
<211> 71
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: plsC-fw<220>
<221> misc_feature
<223> oligonucleotide: plsC-fw - <400> 13 <400> 13
-
<210> 14
<211> 71
<212> DNA
<213> synthetisches Oligonukleotid<210> 14
<211> 71
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: plsC-rev<220>
<221> misc_feature
<223> oligonucleotide: plsC-rev - <400> 14 <400> 14
-
<210> 15
<211> 30
<212> DNA
<213> synthetisches Oligonukleotid<210> 15
<211> 30
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: plsC-check-for<220>
<221> misc_feature
<223> oligonucleotide: plsC-check-for -
<400> 15
cgagattcaa gtagcggcgg aacgggtagc 30<400> 15
cgagattcaa gtagcggcgg aacgggtagc 30 -
<210> 16
<211> 30
<212> DNA
<213> synthetisches Oligonukleotid<210> 16
<211> 30
<212> DNA
<213> synthetic oligonucleotide -
<220>
<221> misc_feature
<223> Oligonukleotid: plsC-check-rev<220>
<221> misc_feature
<223> oligonucleotide: plsC-check-rev -
<400> 16
gttaagtcgt catccggcag cgtattcaac 30<400> 16
gttaagtcgt catccggcag cgtattcaac 30 -
<210> 17
<211> 4487
<212> DNA
<213> Plasmid<210> 17
<211> 4487
<212> DNA
<213> plasmid -
<220>
<221> misc_feature
<222> (1)..(4308)
<223> Plasmid pMT1<220>
<221> misc_feature
<222> (1) .. (4308)
<223> plasmid pMT1 - <400> 17 <400> 17
Claims (7)
- Microorganism strain for the production of low-molecular-weight substances or proteins, containing in its genome a mutation in a gene, which mutation brings about an auxotrophy in the strain, and also a production plasmid encoding at least one enzyme for the production of a low-molecular-weight substance or at least one recombinant protein and also a functional copy of the gene, the chromosomal inactivation of which brings about the auxotrophy, characterized in that the auxotrophy is a nonfeedable auxotrophy and the gene is the plsC gene having SEQ ID No. 3 or a plsC-homologous gene,
wherein a nonfeedable auxotrophy is to be understood to mean that the auxotrophy reduces the growth of the microorganism cell or brings about the death of the microorganism cell and is not supplementable by addition of metabolism-specific precursors, intermediates and/or end products to the growth medium and wherein the plsC-homologous genes are genes which code for a protein having plsC activity and which have a sequence identity greater than 30% in relation to SEQ ID No. 3. - Microorganism strain according to Claim 1, characterized in that the mutation of the gene which brings about the nonfeedable auxotrophy in the strain leads to the inactivation of said gene.
- Microorganism strain according to either of Claims 1 and 2, characterized in that the mutation of the gene which brings about the nonfeedable auxotrophy in the strain leads to the inactivation of the activity of the gene product coded by the gene.
- Method for producing a strain according to any of Claims 1 to 3, characterized in that a temperature-sensitive plasmid is introduced into a microorganism strain which has the plsC gene or a plsC-homologous gene, which temperature-sensitive plasmid has a functional copy of the plsC gene or the homologous gene thereof that is to be mutated or deleted, then the genome of the strain is mutated such that the strain has in the plsC gene or plsC-homologous gene a mutation which leads to a nonfeedable auxotrophy in the strain, and then the temperature-sensitive plasmid is exchanged in said strain at a nonpermissive temperature for a production plasmid, wherein the production plasmid contains at least one gene encoding an enzyme for the production of a low-molecular-weight substance or at least one recombinant protein and also a functional copy of the plsC gene,
wherein plsC-homologous genes are genes which code for a protein having plsC activity and which have a sequence identity greater than 30% in relation to SEQ ID No. 3. - Method according to Claim 4, characterized in that the temperature-sensitive plasmid has a temperature-sensitive origin of replication.
- Method according to Claim 5, characterized in that the cells are exposed, immediately after the transformation with the production plasmid, to a temperature shock at 47-55°C for 30-90 min and the further incubation is then carried out at the nonpermissive temperature of 37-45°C.
- Method for producing low-molecular-weight substances or proteins by means of a microorganism strain in a fermentation medium, which method is characterized in that a microorganism strain according to one or more of Claims 1 to 3 is used and an antibiotic-free fermentation medium is used.
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DK19175665.9T DK3556847T3 (en) | 2015-12-11 | 2015-12-11 | MICRO-ORGANISM STEM AND PROCEDURE FOR ANTIBIOTIC-FREE, FERMENTATIVE MANUFACTURE OF LOW MOLECULAR SUBSTANCES AND PROTEINS |
EP19175665.9A EP3556847B1 (en) | 2015-12-11 | 2015-12-11 | Microorganism strain and method for the fermentative production of low molecular weight antibiotic-free substances and proteins |
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EP3534936B1 (en) | 2016-11-01 | 2020-10-21 | Novo Nordisk A/S | Tolerogenic dna vaccine |
TWI728201B (en) * | 2016-11-01 | 2021-05-21 | 丹麥商諾佛 儂迪克股份有限公司 | Tolerogenic dna vaccine |
US11421239B2 (en) * | 2017-07-21 | 2022-08-23 | Conagen Inc. | Plasmid addiction system to drive desired gene expression |
JP7185006B2 (en) * | 2018-07-06 | 2022-12-06 | ワッカー ケミー アクチエンゲゼルシャフト | Bacterial strains that release recombinant proteins by fermentation |
US11279745B2 (en) | 2019-04-26 | 2022-03-22 | Novo Nordisk A/S | Tolerogenic DNA vaccine |
CN110819672A (en) * | 2019-12-04 | 2020-02-21 | 美亚药业海安有限公司 | Method for preparing cytidine triphosphate by immobilized enzyme method |
CA3217728A1 (en) | 2021-05-20 | 2022-11-24 | Harald Kranz | Microorganism strain and method for antibiotic-free plasmid-based fermentation |
WO2023166132A1 (en) | 2022-03-04 | 2023-09-07 | Gen-H Genetic Engineering Heidelberg Gmbh | Microorganism strain for antibiotic-free plasmid-based fermentation and method for generation thereof |
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EP0251579A3 (en) | 1986-06-24 | 1989-03-22 | Enterovax Research Pty. Ltd. | Non-antibiotic marker system |
RU2091487C1 (en) | 1987-02-27 | 1997-09-27 | Гист-Брокейдс Н.В. | Method of producing bacillus strain containing two copies of dna sequence encoding enzyme exhibiting hydrolase activity |
DE19726083A1 (en) | 1997-06-19 | 1998-12-24 | Consortium Elektrochem Ind | Microorganisms and processes for the fermentative production of L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives |
US6291245B1 (en) * | 1998-07-15 | 2001-09-18 | Roche Diagnostics Gmbh | Host-vector system |
DE10032173A1 (en) * | 2000-07-01 | 2002-01-17 | Degussa | New nucleotide sequences encoding the plsC gene |
DE10109996A1 (en) * | 2001-03-01 | 2002-09-05 | Basf Ag | Process for changing the genome of gram-positive bacteria with a new conditionally negative dominant marker gene |
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PL1945772T3 (en) | 2005-10-06 | 2016-05-31 | Advanced Accelerator Applications S A | Novel selection system |
US8962298B2 (en) * | 2006-05-02 | 2015-02-24 | Butamax Advanced Biofuels Llc | Recombinant host cell comprising a diol dehydratase |
DE102006044841A1 (en) | 2006-09-22 | 2008-04-03 | Wacker Chemie Ag | Signal peptide for the production of recombinant proteins |
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DE502006006800D1 (en) | 2006-09-22 | 2010-06-02 | Wacker Chemie Ag | Process for the fermentative production of proteins |
DE102007021001A1 (en) | 2007-05-04 | 2008-11-06 | Ab Enzymes Gmbh | Expression system for the antibiotic-free production of polypeptides |
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US11046732B2 (en) | 2021-06-29 |
ES2867964T3 (en) | 2021-10-21 |
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CN108463546B (en) | 2022-03-11 |
DK3556847T3 (en) | 2021-05-25 |
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JP6828038B2 (en) | 2021-02-10 |
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